FR2502496A2 - Aerobic bacterial fractions, esp. Corynebacterium catarrhalis - are medicaments with adjuvant, immunostimulant, antibacterial and interferon-inducing activity - Google Patents

Abstract

Active therapeutic fractions obtd. from aerobic bacterial strains are new. The active fractions may comprise the washed entire cells, the delipidated entire cells, insoluble particular extracts of the delipidated entire cells or water-soluble extracts of the delipidated entire cells of the aerobic bacteria. Preferred bacteria are aerobic Corynebacteria, esp. Corynebacterium catarrhalis, which is a new strain of aerobic Corynebacterium . Used as medicaments with adjuvant, immunostimulant, antibacterial and interferon-inducing activity, which may be formulated for intravenous administration or for subcutaneous administration, adsorbed on a mineral gel e.g. calcium phosphate and aluminium hydroxide gels. e.g. 1.365 mg of Al(OH)3 and 1 mg of active fraction per ml. Activity tests show that aerobic Corynebacterium inhibit the growth of Lewis tumours, give 95-100% protection when 5 x LD50 and 80-100% protection when 10 x LD50 doses of E-coli, K-pneumoniae or L. monocytogenes are administered to mice and protect mice against Listeria monocytogenes infections. The fractions are used to increase resistance to bacterial and fungal infections in immune-deficient patients, protect patients having certain malignant tumours against infections and to treat patients undergoing immuno-suppressive treatment such as intensive chemotherapy or radeotherapy.

Description

 This Addition relates to perfections to fractions obtained from aerobic bacteria conforming to the main patent.

 According to the main patent, adjuvant, immunostimulatory and antibacterial activity fractions are obtained from strains of bacteria subjected to delipidation treatment and, more particularly, from whole cells of delipidated aerobic bacteria, such as those of a strain of newly isolated aerobic Corynebacterium, named Coryne bacterium catarrhalis, deposited on February 21, 1980, under No. 116, in the National Collection of Microorganisms held by INSTITUT PASTEUR.

 When the delipidated whole cells are subjected to a gentle extraction treatment by milling and homogenization in an aqueous medium, in the presence or absence of a nonionic detergent, such an extraction treatment makes it possible to obtain an insoluble particulate fraction which is in addition to the adjuvant, immunostimulatory and antibacterial properties of the delipidated whole cells, it also has interferon inducing properties; on the other hand, the water-soluble fraction also obtained following said extraction treatment, if it exhibits interferon-inducing properties, does not exhibit the adjuvant, immunostimulatory and antibacterial properties that the insoluble particulate fraction in question possesses in addition.

The purpose of this Addition is to provide enhancements to the active fractions in accordance with the main patent, in particular by allowing their administration with a very satisfactory level of efficacy, not only intravenously, but also subcutaneously, including the water-soluble fraction which has been shown to be inactive intravenously in the treatment of bacterial infections, particularly in the treatment of
Listeria monocytogenes in mice.

The object of this Addition is agents
anti-infectious agents, characterized in that they consist of active fractions of aerobic bacteria, in particular of aerobic Corynebacteria and, specifically, of
Corynebacterium catarrhalis
According to an advantageous embodiment of the anti-infective agents according to the invention, their active fractions consist of washed whole cells put in a form allowing their intravenous administration.

 According to another advantageous embodiment of the anti-infective agents according to the present Addition, the active fractions consist of delipidated whole cells put into a form allowing their intravenous administration.

According to yet another advantageous embodiment of the anti-infective agents according to the present invention.
In addition, the active fractions consist of insoluble particulate fractions extracted from whole cells delipidated from aerobic bacteria, put into a form allowing their intravenous administration.

 According to an advantageous embodiment of the anti-infective agents according to the present Addition, they are put in a form allowing their administration subcutaneously and their active fractions are constituted by whole cells washed or by whole cells delipidated aerobic bacteria, or by particle fractions obtained by gentle extraction of delipidated whole cells or by water-soluble fractions obtained by gentle extraction of whole cells delipidated.

 According to a preferred embodiment of this embodiment, the active fractions of the anti-infective agent according to the present invention, are made capable of being administered subcutaneously, by adsorbing them on a mineral gel.

 In accordance with this Addition, the inorganic gel on which the active fractions of aerial bacteria are adsorbed to allow their administration subcutaneously is selected from the group which comprises, in particular, calcium phosphate gels and gels. aluminum hydroxide.

 As is known, different species of Coryr anaerobic bacteria have been shown to be potent stimulating agents of the reticuloendothelial system. In addition, among Corynebacterium parvum and Corynebacterium granulosum, Corynebacterium granulosum has been shown to increase resistance to various bacterial infections. As indicated in the main patent, the fractionation of whole cells of anaerobic Corynebacteria gave rise to fractions which show the immunoadjuvant activities of the intact bacterium. It has been demonstrated by one of the inventors of the subject matter of this Addition that an insoluble particulate fraction isolated from Corynebacterium granulosum, referred to as the P40 fraction, exhibits potent immunostimulatory and adjuvant activity.

 In contrast, a number of authors have demonstrated that aerobic Corynebacteria either lack immunostimulatory and adjuvant activity, or exhibit very low activity of this type.

As stated in the main patent, the Insurers have isolated a new species of aerobic Corynebacterium which they have referred to as Corynebacterium.
Catarrhalis and from which they have isolated fractions from delipidated whole cells, which fractions have important activities as interferon inducer.

 To demonstrate the possibilities of therapeutic use of fractions thus isolated, for the prevention or treatment of infections in patients with an immune deficiency, the inventors have continued their research to determine whether the whole cells or fractions of Corynebacterium catarrhalis could increase the resistance of mice to Listeria monocytogenes.

 As is known, Listeria monocytogenes is an aerobic non-sporulated germ that is the agent of an infectious disease common to humans and animals, listeriosis, which can reach particularly serious forms.

The inventors have demonstrated the anti-infectious action exerted by Corynebacterium catarrhalis by demonstrating the protective action exerted by the latter against the infection induced by Listeria monocytogenes in mice, by proceeding as follows
I. Preparation of the anti-infective agent
) Culture of bacteria
Corynebacterium catarrhalis was cultured in a 50-liter investigational fermenter. The culture medium consisted of meat broth supplemented with 5 g / liter of Bactopeptone and 2 g / liter of yeast extract. The pH was adjusted to 7.4. The bacteria were harvested by centrifugation of the culture at the beginning of the stationary phase. The bacteria were washed twice by resuspending in distilled water and centrifuging.

Washed bacteria designated under the acronym
BL were used either as such or after being delipidated according to the method of AEBI & Alia, mentioned in the main patent; delipidated bacteria are referred to as BD.

2) Fractionation of bacteria
Whole cells delipidated from Corynebacterium catarrhalis were fractionated using the method of D.MIGLIORE-SAMOUR & Alia mentioned in
Main patent. The particulate fraction precipitated from an ammonium sulfate solution at 40% saturation is designated CBAp4O. The water-soluble fraction contained in the supernatant after precipitation with ammonium sulfate at 70% saturation is symbolized by the acronym CBA-LS. CBAp40 and CBA-LS were dialyzed until exhaustion against distilled water. The
CBAp40 was stored at -200C as a wet product, and CBA-LS as lyophilizate.

II.-Adsorption on mineral gels
Whole cells as well as insoluble particulate fractions and water-soluble fractions were adsorbed on calcium phosphate gel or aluminum hydrate gel using one of the following methods: 1. adsorption on gel calcium phosphate
a) adsorption on calciun phosphate gel
was carried out by addition, with stirring,
a determined volume of bacteria suspension
or CBAp40 or CBA-LS solution in
0.07 M Na 2 HPO 4, with an equal volume of 0.07 CaCl 2
The pH of the mixture was then adjusted to
neutrality by addition of 0.1 N NaOH

(b) The product may also be adsorbed
subjected to experimentation, on a gel of phos
preformed calcium pellet that is prepared from the
following way
100 ml of dihydrogen phosphate solution are mixed
0.07 M sodium and 100 ml solution of
0.07 M CaCl2. The pH is adjusted to 7 by addition
0.1N NaOH. The gel forms is sedimented by
centrifugation and washed twice with a solution
of isotonic NaCl.

To this preformed and washed gel, the product is added
to adsorb that can be the solution of CBA-LS
in isotonic NaCl or a suspension of
bacteria or CBAp40 in isotonic NaCl.

The mixture is stirred for 30 minutes with a
magnetic stirrer so as to allow
adsorption of the product. This gel is centrifuged
and washed with isotonic NaCl.

2. Adsorption on aluminum hydroxide gel
The adsorption on aluminum hydroxide gel was obtained by mixing quantities of "ALHYDROGEL" (Superfos Export Co.) and the product under experiment, in proportions such that the final mixture contained 1.365 mg. A (OH) 3 / ml and 1 mg of the product subjected to the experiment / ml.

III.-Doses and routes 1. Female SWISS mice were used as animals.
from 17 to 19 g. The animals were submitted to the diet
laboratory and received water ad libitum.

2. The products submitted to the experiment were injected
at a dose of 400 μg (dry weight) in physiological serum
cal.

a) Injections were made intravenously
(IV) for non-adsorbed products,
in a volume of 0.2 ml,
(b) the injections were made
cutaneous (SC) for adsorbed products, under a
lume of 0.4 ml.

3. Control mice were injected
- or physiological serum intravenously,
- either a calcium phosphate or hydroxide gel
of aluminum not containing the products subject to
subcutaneous experimentation1.

IV.- Infection
Mice were infected with Listeria monocytogenes obtained by culture for 18 hours of strain 1150, by intravenous injection at an appropriate dilution (3-4 MLD); the injection took place six days after the mice had received one of the products subjected to the experimentation.

 This dose of Listeria monocytogenes produced 100 deaths in control mice in 24 to 96 hours. The deaths were recorded for ten days.

 In some cases, the mice were again infected 75 days after the first infection by intravenous injection of 20 MLD of Listeria monocytogenes.

V. Results
The results obtained by previously treating mice intravenously, either with BL or with BD, are shown in Table I below.

TABLE I
Protective effect of the prior intravenous treatment of mice with whole cells, either washed (BL) or unfolded (BD), Corynebacterium catarrhalis, against Listeria monocytogenes infection

<SEP> Rate <SEP> of
<tb> Products <SEP> Deaths <SEP> occurring <SEP> at <SEP> day <SEP> death
<tb><SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
<tb> BL <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 2 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 2/8
<tb> BD <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/10
<tb> Controls <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10/10
<Tb>
It is apparent from Table I above that 100% mortality was observed in the control mice within 24 hours after infection with Listeria monocytogenes.

 In contrast, a high degree of protection (75-80s of the mice were protected) was obtained when the mice had been stimulated by intravenous infection, either BL or BD, five days before being infected with Listeria monocytogenes. . These results clearly show that pretreatment of intravenous mice by whole cells of Corynebacterium catarrhalis can effectively increase their resistance to Listeria monocytogenes.

 The results obtained when the mice are pre-treated subcutaneously, either with BL or with BD previously adsorbed either on a calcium phosphate gel or on an alumina hydroxide gel, are collected in Table II which follows.

TABLE II
Protective effect against Listeria monocytogenes infection of a subcutaneous pretreatment of mice, either by whole cells washed (BL) or delipidated (BD) of Corynebacterium catarrhalis adsorbed or on a calcium phosphate (PC) gel, either on an alumina hydroxide gel (HA).

Products <SEP> Deaths <SEP> occurring <SEP> at <SEP> days <SEP> Rate <SEP> of <SEP> deaths
<tb><SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 8 <SEP> 9 <SEP> 10
<tb> BL <SEP> (PC) <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/10
<tb> BL <SEP> (HA) <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/10
<tb> BD <SEP> (PC) <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/10
<tb> BD <SEP> (HA) <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/10
<tb> Serum
<tb> physiological <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10/10
<tb> (PC) <SEP> alone <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 4 <SEP> 4 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 8/8
<tb> (HA) <SEP> alone <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 4 <SEP> 2 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 6/8
<Tb>
In the experiment reported in Table II above, it was also verified that the mortality of Listeria monocytogenes-infected mice in 24 hours was 100%. The results presented in this Table show, moreover, that the pre-treatment of mice subcutaneously by adsorbed products is extremely effective because it makes it possible to obtain 100% protection against the infection of animals with Listeria monocytogenes. .

 In contrast to this, the death of pretreated mice subcutaneously either with calcium phosphate salt or with aluminum hydroxide gel, in the absence of bacteria, was only delayed. after infection with Listeria monocytogenes, compared with the death of control mice infected the same way.

The protective power of the CBAp40 and
CBA-I against Listeria monocytogenes infection was measured in mice. The activity of CBAp40 and
CBA-LS was determined during experiments whose results are summarized in Table III below: TABLE II
Protective effect against mouse infection by Listeria monocytogenes a) pre-treatment of mice intravenously with either CBAp40 or CBA-LS, b) pre-treatment of mice subcutaneously with either CBAp40 or by CBA-LS
adsorbed either on calcium phosphate (PC) or on hydroxide
aluminum (HA)

<SEP> Deaths <SEP> occurring <SEP> at <SEP> days <SEP> Rate <SEP> of <SEP> deaths
<tb> Products <MS> Pathway <SEP> 1 <SEP> 4 <SEP> 5 <SEP> 6 <SEP> 7 <SEP> 9 <SEP> 10
<tb> (CBAp40) <SEP> iv <SEP> 0 <SEP> 0 <SEP> 2 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 2/10
<tb> (CBAp40) <SEP> (PC) <SEP> sc <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 1 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 1/8
<tb> (CABp40) <SEP> (HA) <SEP> sc <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0/8
<tb> (CBA-LS) <SEP> iv <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 10/10
<tb> (CBA-LS) <SEP> (HA) <SEP> sc <SEP> 0 <SEP> 1 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 1 / 10
<tb> Serum <SEP> iv <SEP> 0 <SEP> 10 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 10/10
<tb> physiological
<Tb>
The results that are gathered in this Table
III show that the injection of Listeria monocytogenes to the control mice resulted in 100% mortality of the animals. The pretreatment of the mice intravenously or subcutaneously by CBAp40 provided protection for 80% of the animals. The anti-infectious activity of CBAp40 adsorbed on a calcium phosphate or alumina hydroxide gel allowed to protect .90-100% of animals against Listeria monocytogenes infection.

Intravenous injection of CBA-LS did not exert any protective effect against
Listeria monocytogenes. In contrast, injection of CBA-LS adsorbed on calcium phosphate or alumina hydroxide gel subcutaneously provided effective protection against infection with Listeria monoctogenes.

The results of the experiments summarized in Tables I, II and III above clearly show that both whole cells and Cor bacterium catarrhalis fractions are capable of significantly increasing the resistance of the infection-infected host.
Listeria monocytogenes, whether injected intravenously alone or subcutaneously as an adsorbed product or on calcium phosphate; or on alumina hydroxide in gel form.

 Protection was obtained with intravenously administered Corynebacterium catarrhalis bacteria and fractions, except for CBA-LS which did not provide protection by this route.

 Protection was also effective when products adsorbed on calcium phosphate or alumina hydroxide, including CBA-LS, were administered subcutaneously. In addition, it was a jack: that the injection of a gel either of calcium phosphate or of alumina hydroxide in the absence of the active products subjected to experimentation, did not protect the mice. Listeria monocytogenes infection.

 It seems that the mechanism which increases the resistance to Listeria monocytogenes and which is conferred on the mice by whole cells as well as by the delipidated cells of Corynebacterium catarrhalis, and also by CBAp40 and by CBA-LS, is not the same as that whereby Corynebacterium parvum, which is an anaerobic Corynebacterium, acts to increase the resistance of mice to Listeria monocytogenes: indeed, the increase of resistance to Listeria monocytogenes ensured by Corynebacterium parvum has been attributed to the activation of fixed and free macrophages. . However, this explanation does not appear to be correct for whole cells and fractions of Corynebacterium catarrhalis, because these do not increase the activity of the reticuloendothelial system, as is the case for Corynebacterium parvum.

In any case, and whatever the mechanism of action, we obtain drugs that increase the resistance to infection of individuals with immune deficiency, whether they are patients presenting certain malignant tumors that increase their susceptibility to infections by bacteria and
fungi, or patients receiving immunosuppressive therapy such as intensive chemotherapy. or radiotherapy.

Claims (7)

 1.Anti-infectious drugs characterized by being active fractions of aerobic bacteria, especially aerobic Corynebacteria and specifically, Corynebacterium catarrhalis according to claim 8 of the main patent  2. Anti-infective drugs according to Claim 1, characterized in that their active fractions consist of washed whole cells put into a form allowing their intravenous administration.  3. Anti-infective drugs according to Claim 1, characterized in that their active fractions consist of whole delipidated cells put into a form allowing their intravenous administration.  4. Anti-infective medications according to Claim 1, characterized in that the active fractions consist of insoluble particulate fractions extracted from whole cells delipidated from aerobic bacteria, put into a form allowing their intravenous administration.  5. Anti-infective drugs according to claim 1, characterized in that they are put in a form allowing their administration subcutaneously and in that 14 active fractions are constituted by washed whole cells or by whole cells delipidated aerobic bacteria, or by particulate fractions obtained by gentle extraction of whole cells delipidated or still by water-soluble fractions obtained by gentle extraction of whole cells delipidated.  Anti-infective drugs according to Claim 5, characterized in that the active fractions of said anti-infective medicament are made suitable for administration subcutaneously by adsorbing them on a mineral gel.  7. anti-infectious drugs according to Reven dication 6, characterized in that the mineral gel on which the active fractions of aerobic bacteria are adsorbed to allow their administration subcutaneously is selected from the group which comprises in particular the phosphate gels of calcium and aluminum hydroxide gels.  80- A process for the preparation of subcutaneously administrable anti-infective drugs according to claim 6 or claim 7, characterized in that the adsorption of the whole cells delipidated or not, or insoluble particulate fractions or water-soluble fractions which The active fractions of said medicaments on calcium phosphate gel are made by adding, with stirring, a determined volume of 0.07 M Cacao2 to an equal volume of suspension of said insoluble particulate or fractional solution cells or fractions. water-soluble, the pH of the mixture then being adjusted to neutrality by the addition of 0.1 N NaOH.  9 - Process for the preparation of anti-infective drugs suitable for subcutaneous administration according to claim 6 or claim 7, characterized in that: during a first step, a preformed gel is prepared by volume-to-volume mixing a 0.07 M disodium phosphate solution and a 0.07 M CaCl 2 solution, adjusting the mixture to about pH 7 by addition of 0.1 N NaOH, sedimentation of the formed gel and washing with a chloride solution isotonic sodium; during a second step, the active fraction to be adsorbed-suspension of whole bacteria or insoluble particulate fractions or solutions of water-soluble fractions is added to the preformed gel, washed, and then the mixture is stirred for a sufficient time to allow the adsorption of the product on the gel, after which the finished product is centrifuged and washed with an isotonic solution of NaCl.  100- A process for the preparation of anti-infective drugs suitable for subcutaneous administration according to claim 6 or claim 7, characterized in that the active fractions of said medicaments - suspension of whole bacteria or insoluble particulate fractions or fraction solutions water-soluble - are adsorbed on aluminum hydroxide gel by mixing aluminum hydroxide gel and said active fractions in proportions such that the final mixture contains 1.365 mg of Al (OH) 3 / ml and 1 mg of active fraction / ml.