CS270410B2 - Method of biologically active substance production with immunostimulating effect - Google Patents

Abstract

1. Immunomodulating medicament of biological origin, of which the active principle is formed by the products of lysis or a fraction of these products of bacteria lysed by bacteriophagic route, characterised in that it contains, as active principle, the sterile lysis filtrate of at least two bacteria lysed by homologous bacteriophagic route ; the bacteria chosen being Klebsiella pneumoniae and Escherichia coli.

Description

(57) The medicinal product affecting the immune response consists of the lysis products or fractions of the lysis products of bacteria of the genus Escherichia, Streptococcus, Klebsiella, Staphylococcus or others using bacteriophages. A first inoculation of the culture broth with the inoculum of a bacterium of one of the genera is carried out, during the growth phase, the bacteria of said genus are infected with a frequency of 0.01 to 0.1 by introducing their respective phages. per ml. A second inoculation of the culture broth is then carried out with an inoculum of bacteria of one of the genera mentioned. During the growth phase of the second genus, the bacteria of the second genus are infected with an infection rate of 0.01 to 0.1 by introducing their respective phages. The development of lysis of these second genus bacteria is monitored to a specified degree of 10 to 1000 bacteria per ml. Finally, the non-lysed bacteria are removed and the lysis filtrate obtained from both genera of bacteria is collected.

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The invention relates to a process for the preparation of immunomodulatory drugs of biological origin.

Many preparations are known to modulate the immune response by fractions or extracts of lysed mirobial cells. Particular mention may be made of the products described in French patents 2,374,042, 2,396,018 and 2,426,470.

These lysates or extracts are usually obtained by physical, chemical or less often enzymatic processes. Although most of these formulations have some immunomodulatory activity in animals, their therapeutic efficacy in humans is only the subject of a number of studies (Immunopharmacologie clinique. Realités et Perspectives G. RENOUX. Pharmacologs et thérapeutique. R.P. 1982, 32, 41-42).

It was therefore important to find drugs that would be a weapon applicable to both human and veterinary medicine whenever there was a need to renew or strengthen immune defense. <

A new preparation method using bacteriophage has been found. Bacteriophage, which is an excellent agent for bacterial lysis, has been used to produce immunomodulatory preparations with different genera and species of bacteria. This bacterial lysis technique using bacteriophage is not limited as to the genus or species of bacteria. This method has made it possible to obtain drugs excellent in their ability to modify the immune system, prepared from one or more species of bacteria or a mixture of these species. In particular, these pharmaceuticals have the ability to induce the production of interferons.

These immune response medicaments, the active ingredient of which is a lysate product or a fraction of lyses of bacteria by a bacteriophage according to the invention, contain as active substance lysate products of at least two bacteria by the corresponding bacteriophages.

Bacteria include, for example, Escherichia coli, Streptococcus faecalis, Klebsiella pneumoniae, Staphylococcus aureus. Of particular interest is the preparation obtained using the species of Klebsiella and Escherichia.

The obtained drug may be combined with an antibiotic preparation such as tetracyclines, antiviral such as synthetic nucleosides blocking the synthesis of viral nucleic acid, antifungal such as buclosamide, or antiparasitic such as chloroquine. ,

The medicament obtained by the method of the invention may also be combined with other pharmaceutical products and compositions, such as an anti-tumor agent (cytostatics), an immunosuppressive agent (antilymphocytic immunoglobulins), a mitosis suppressant such as folic acid derivatives, or an anti-inflammatory such as indomethacin.

The medicament prepared by the method of the invention may be administered in various forms by intradermal, subcutaneous, sublingual, topical, oral, parenteral or bronchial. If desired, the medicament is mixed with a suitable vehicle and used in the form of an aqueous or alcoholic solution or suspension in an oily vehicle, or in the form of a lyophilized or aerosol or ointment.

The dosage of the drug produced by the method of the invention may vary within wide limits depending on the composition of the drug, the prescribed indication, the form of administration and the age of the subject being treated. The determination of appropriate doses in influencing the immune response is left to the discretion of the physician or veterinarian as is conventional.

For example, daily dosages for the treatment of chronic or recurrent respiratory tract infections, for the prevention of postoperative complications or for the prophylaxis of urinary tract infections may range from 1 to 50 ml, depending on the concentration of the immunomodulatory agent.

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The medicament produced by the method of the invention is well tolerated; its toxicity is low, as shown by the OL ^ q test, which could not be completed orally due to the gastric capacity of the test animals (mice, rats).

In particular, the medicaments obtained by the method according to the invention are indicated in the treatment of immune response disorders, in the prevention or treatment of serious bacterial, viral, fungal or parasitic infections, as well as in rheumatology and in the scarring and protection of wounds.

The method for producing a biologically active substance having an immunostimulatory effect, which consists of the lysis products or the fraction of the lysis products of bacteria of the genus Escherichia, Streptococcus, Klebsiella, Staphylococcus or the other using bacteriophages according to the invention is that , during the growth phase, the bacteria of said genus are infected with an infection rate of 0.01 to 0.1 by introducing their respective phages, monitoring the lysis development to a predetermined level of 10 to 1000 bacteria per milliliter, then inoculating the culture broth a second time bacteria of one species, during the growth phase of the bacteria of the second genus, the bacteria of the other genus are infected with an infection rate of 0.01 to 0.1 by introducing their respective phages, monitoring the development of lysis of these bacteria of the second genus until a degree of 10 to 1 000 bacteria per milliliter, finally remove the non-lysed bacteria and collect the filtrate obtained from both genera. Removal of non-lysed bacteria and bacterial residues is accomplished by methods known to those skilled in the art, such as filtration, centrifugation, and the like. The process according to the invention can advantageously be carried out by successive use of Klebsiella and Escherichia.

The following are exemplary embodiments that illustrate the invention without limiting it.

Example 1

Preparation based on one bacterium of the genus Escherichia

The bacterium used is of the genus Escherichia, coli species, serotype 0119: В 14, registered by the Pasteur Institute in Paris under number 62.23.

The colifage belongs to the morphological group (Nomenclature of the Subcommittee on Taxonomy of Bacteriophages of the International Committee for Taxonomy of Virus IAMS). It has an isometric head, a very short tail that is hard to detect, a sleeve and end plate that are not visible. On agar, the backyards of the skis have a diameter of 1 to 1.5 mm, a round form, regular edges, a cloudy center, and converging clones ^{r 7} .

The culture medium contains per liter: bovine extract 2.5 g, peptone 4.1 g, sodium chloride 7.0 g, the rest redistilled water.

The soil is inoculated with an inoculum of bacteria that are in an exponential growth phase. The initial density of the culture is 1 .mu.g bacteria per ml. Incubation is performed at 37 ° C with gentle agitation.

In the middle of the exponential growth phase, i.e. approximately 60 minutes after inoculation, the bacteria are infected with their respective phages. Infect 0.02 fold.

Ski development is monitored using a cell calibrated in advance. When less than 1,000 bacteria per ml are used, sterile filtration is performed by passing through a filter membrane having a porosity close to 0.22 µm. The ski filtrate is already an imonomodulatory preparation.

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Example 2

Preparation based on one bacterium of the genus Streptococcus

The bacterium of the genus Streptococcus, faecalis species, variant of zymogenes, registered by the Pasteur Institute in Paris under number 53.152 shall be used.

The phage belongs to the morphological group (nomenclature of the Taxonomy of bacteriophages subcommittee of the International Community for Taxonomy of Virus IAMS). Its head is isometric, the tail is long, the case is visible and the end plate is complex. On agar, the backyard skis are round, iiiohsi наг, B Hint on average, jtijioh's edges are regular to strad clear. There are no ^{r T} clones or only one.

The culture medium contains per liter: bovine extract 0.3 g, tryptase 1.0 g, dextrose 1.0 g, sodium chloride 0.5 g, the rest redistilled water.

The soil is inoculated with bacteria in stationary phase. The initial density is 1.10 ⁷ bacteria per ml. Store in the dark at 37 ° C with gentle stirring.

At the beginning of the exponential growth phase, i.e. approximately 40 minutes after seeding, the bacteria are infected with their respective phages. The infection rate is 0.1.

Ski development is monitored using a cell counter. When less than 10 bacteria per ml remain, the culture is sterile centrifuged to remove lysogenic bacteria and non-lysed bacterial cells. The rest is an immunomodulatory preparation.

Example 3

Preparation with the gradual use of Klebsiella and Escherichia

The bacterium of the genus Klebsiella, a species of pneumoniae, is registered by the Institute of Pasteur in Paris under chimney 50.5.

Further details on the bacterium of the genus Escherichia are given in Example 1.

The Klebsielle phage belong to the morphological group (Nomenclature of the Subcommittee on Bacterariophage Taxonomy of the International Committee for Taxonomy of Virus IAMS). The head has a lkosaedrickou, tail short, hard to detect. The housing and end plate are not detectable. On agar, the backyard skis have a diameter of 0.5 to 3 mm and have a very irregular shape with melting edges and a clear center. Clones ^{r T} are sparse.

The properties of colifag are described in Example 1.

The culture medium contains per liter: beef extract 3 g, popton 5 g, sodium chloride 7 g, the rest redistilled water.

The soil is inoculated with Klebsielly pneumoniae inoculum in the stationary growth phase. The initial density is 1.10 ⁷ bacteria per ml. The culture is heated to 37 ° C with gentle stirring.

Towards the end of the latent phase, i.e. approximately 60 minutes after seeding, the bacteria are infected with their respective phages.

The infection rate is 0.03.

The development of the ski is monitored using a cell counter. As soon as less than 10 bacteria per ml remain, the soil is inoculated with the Escherichia inoculum in the stationary growth phase. Initial density

CS 270 410 B2 of this new culture is 5.10? Escherichia per ml

Towards the end of the latency phase, which is 20 minutes after the second inoculation under these experimental conditions, the bacteria are infected with colifages. The infection rate is 0.1.

The development of the ski is monitored using a cell counter. When less than 10 µm Escherichia per ml remains, the culture is allowed to pass through a 0.22 µm sterilizing filter membrane. The obtained lysate filtrate obtained from two bacteria, Klebsiella pneumoniae and Escherichia coli, is an immunomodulatory preparation.

Example 4

Preparation and use of one bacterium of the genus Staphylococcus

A bacterium of the genus Staphylococcus, aureus species, serotype 1 is registered by Pasteur's mouth in Paris under number 65.6.

The respective Staphylococca phage belong to the morphological group (subcommittee Nomenclature of the bacteriophage taxonomy International Committee for Taxonomy of Virus.de IAMS). It has an octahedral head and the tail is completely covered with a holster and a complex end plate. On agar, the backyard skis have a diameter of 1 to 2 mm, are round and regular, their center is clear. There are no ^{r T} clones.

The bacteria are decomposed into solid soil containing per liter: protein peptone No. 3 Oifco 10.0 g, dextrose 1.2 g, soluble starch 6.0 g, sodium chloride 3.0 g, sodium phosphate 2.0 g, Bacto gelatin 13 0 g, Bacto agar 6.0 g, distilled water qs

After 24 hours, the microorganisms that formed the dense bacterial carpet are sterile collected, numbered and resuspended in soil containing per liter: bovine extract 2.5 g, peptone 4.1 g, sodium chloride 7.0 g, redistilled water qs so to a density of 5.10 ⁶ bacteria per ml.

At the same time, corresponding phages are introduced into the soil. The infection rate is 0.01.

• 2

The development of the ski is monitored using a cell counter. Once less than 10 bacteria per ml are left, membrane filtration is performed with a porosity of approximately 0.22 µm. The lysate filtrate already constitutes an immunomodulatory preparation.

Pharmacological Study:

The effects of the formulations of the invention in animals were demonstrated by the formulation corresponding to the Escherichie coli ski filtrate obtained as described in Example 1. The filtrate was lyophilized, then redissolved in sterile distilled water. The concentration factor of this new solution was expressed compared to the formulation prior to lyophilization.

Protective effect against acute bacterial infection

This test, demonstrating an overall simulation of the immune system, was performed with infectious microorganisms different from those used in the manufacture of the medicament of the invention, in order to make it clear that the preparation is not effective by its potential vaccine or direct action as bacteriophage but by stimulating immune defense.

Two microorganisms, Klebsiella pneumoniae, bacteria with intracellular development, and Staphylococcus aureus, bacteria with extracellular development, were tested.

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Bacteria causing infection were given to groups of 10 female Swiss mice weighing 18 g on day D _{Q by} intraperitoneal injection of 0.5 ml.

The immunomodulatory preparation was injected the day before the infection was 0. The product was administered intraperitoneally or intravenously in order to avoid a possible positive result from the direct effect of the product on microorganisms at the time of injection.

In order to detect the virulence of the infectious microorganism, which can vary within wide limits from one experiment to another, its lethal dose was determined in each test.

The protective effect of the preparation according to the invention against the actual bacterial infection is documented in the following Table I.

Tabulkal '

bacteria application preparation surviving (%) causing infection ^DL 50 way volume □ avKa concentration factor after application (n = 10) control (n = 10) P

Klebsiella 10 ' ² i.p. 1.0 ml 1 X 0 10 NS 10 ” ³ i.p. 1.0 ml 1 X 70 30 0.050 ⁴ ' ⁴ i.p. 1.0 ml 1 X 100 ALIGN! 20 May 0.001 10 ' ² i.v. 0.5 ml 1 X 20 May 10 NS 10 ~ ³ i.v. 0.5 ml 1 X 80 20 May 0,025 10 “ ⁴ i.v. 0.5 ml 1 X 100 ALIGN! 20 May 0.001 Staphyloco- 10 ' ² i.p. 1.0 ml 1 X 10 10 NS ccus aureus 10 ' ³ i.p. 1.0 ml 1 X 80 10 0.001 10 ' ² i. v. 0.5 ml 1 X 10 20 May NS 10 ' ³ i. v. 0.5 ml 1 X 100 ALIGN! 30 0.001

Stimulation of phagocytic effect of macrophages

The Halper test (Stiffel С., 1958. Phys Phys. 58, 911-949) consists in injecting mice intravenously with colloidal carbon particles, which are mainly coagulated by reticuloendothelial cells of the liver and spleen. The speed of their purification corresponds to the phagocytic effect. They are frustrated by the determination of the free particles remaining in the blood photometrically against time. LPS, a lipopolysaccharide endotoxin extracted from endobacteria, is a potent activator of macrophages.

The test is performed as follows:

- groups of 5 female Swiss mice with an average weight of 24 g were administered intraperitoneally with 0.6 ml of Escherichia coli filtrate or LPS (lipopolysaccharide) solution at a concentration of 0.625 µg / ml.

- after 24 hours, the mice are injected intravenously with 0.12 ml of a colloidal carbon suspension containing 32 mg / ml carbon in 4 gelatin.

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- within 12 minutes, 20 µl of blood from the retro-orbital cavity is collected every 2 minutes from the mice. This collected blood is immediately diluted with 4 ml of 1% Na ₂ CO 3. The solution is then measured photometrically.

the knotting of the oxonocyanate is carried out at 620 nm,.

- the colloidal carbon concentration in the blood at time t of the experiment is determined by subtraction on a pre-calibrated handle. The phagocytic index ee is calculated according to the following pattern. ip. .12Β.9 ₂ -: - ^ 0-5ι ' ^T z - ^T i where C ₂ and Cj represent the concentration of colloidal carbon in the blood at the last and first blood collection, T? and T 1 represent the moment of these subscriptions.

The results summarized in Table II demonstrate that the effect of mouse peritoneal macrophages is stimulated after intraperitoneal administration of the formulation.

Table II preparation (E. lyophilized lysate filtrate using bacteriophage) unvaccinated broth (negative control)

LPS (positive control) application. injection volume I.P.

0.6 ml 0.028 - 0.003

0.6 ml 0.012 - 0.001

0.6 ml; 0.025 - 0.002

Ability to induce serum interferon (IFN serum) production *

IFN is a glycoprotein having a molecular weight of 10,000 to 20,000 daltons. It has many immunomodulatory effects, in particular it stimulates the activity of Natural Killer (NK) cells. Its mode of action is still under investigation. His involvement in anti-virus defense is clearly proven.

Verify hours after administration of 8 female 25 g Swiss mice, 0.3 ml of blood is collected from each into a non-heparinized tube. Serum IFN is determined by the classical method of measuring the cytopathic effect of Vesicular Stonatitis Virus (VSV) infecting mouse L cells cultured for 24 hours in the presence of serum to be tested. (Havell E.A. and Vilcek J., Antimicrob. Agents Chemotherapy 2, 476-484, 1972). The induced protective effect of IFN is compared to that of IFN in mice, an international standard from the National Institute of Health, Bethesda, Sp.st. am. . .

The preparation induces serum IFN in mice two hours after its intraperitoneal, intravenous or oral administration, as shown in Table III.

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Table III

way product volume concentration factor U.I. IFN / ml i.p. preparation 1 * 5 ml 80 1058 placebo 1 * 5 ml 80 0 i. v. preparation 0 * 7 ml 80 950 placebo 0 * 7 ml 80 0 after. preparation 1 * 0 ml 1000 1200 placebo 1 * 0 ml 1000 0

Human activity was tested with the formulation of Example 3 applied first to a volunteer * then to a patient.

A double-blind, conducted in 15 volunteer patients, made it possible to demonstrate that oral administration of the preparation caused a significant increase in cell-mediated immune responses after 10 days. .

A second experiment conducted in 25 patients showed that a single oral administration of different doses of the product allowed the induction of serum lymphokine type MIF - LIF after 24 hours. This induction is dose dependent. ’

Several attempts made it possible to demonstrate the stimulation of IFN production by lymphocytes and monocytes by human cell separation.

IFN * whose production was induced by. * Was characterized using anti-IFN * antisera or. Its production is dose-dependent.

Several clinical trials are described below as examples.

Prevention and treatment of chronic or recurrent respiratory tract infections.

Two groups of 25 patients affected by chronic or recurrent bacterial bronchopulmonary infections were treated by oral administration in one group of 15 ml of the preparation according to the invention for 20 days per month for six months, in the other group of classical antibiotics. The reduction in the number of serious infectious events over the six months duration of the experiment is considerably greater in the group treated with the preparation of the invention than in the group treated with classical antibiotics.

Prevention of postoperative infections

Oral administration of 20 ml of the formulation made it possible to significantly reduce infectious postoperative complications in 106 patients, compared with a reduction in 72 other patients treated with classical therapy.

Prevention and treatment of urinary tract infections

Four authors studied the effect of the above preparation in patients affected by chronic or recurrent urinary tract infection to a clinical extent. Oral administration of 15 ml per day * for 20 days per month within three months allowed a significant reduction in the frequency and duration of infectious events as well as! consumption of anti-infective drugs during the six months of observation compared to the six months prior to treatment.

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In all experiments, the tolerance of the product was considered excellent, both by the physician and by the patient (volunteer or ill).

Claims (2)

SUBJECT OF THE INVENTION (1) a method of producing a biologically active substance in an immunostimulating effect which produces lysis products or fractions of these lysis products by bacteria using bacteriophages, the bacteria being of the genera Escherichia, Streptococcus, Klebsiella, Staphylococcus or other, characterized in that the first inoculation is carried out culture broths by inoculating bacteria of one of the genera mentioned above, during the growth phase, the bacteria of that genus are infected with an infection rate of 0.01 to 0.1 by introducing their respective phages, and the lysis development is monitored up to a predetermined degree of 10 to 1,000 bacteria per milliliter , then proceed to the second inoculation of culture broth with an inoculum of bacteria of one genus, during the growth phase of the bacteria of the second genus, the bacteria of that second genus are infected with an infection rate of 0.01 to 0.1 by introducing their respective phages; up to set In this step, 10 to 1000 bacteria per milliliter are finally removed, and the lysate filtrate obtained from both genera of bacteria is removed. 2. A method according to claim 1, characterized in that the bacteria of the genera Klebsiella and Escherichia are subjected to lysis by the respective bacteriophages.