FR2810337A1 - New microorganisms expressing heterologous gene, useful e.g. for detoxification, have high survival rates in stomach and colon - Google Patents

Abstract

Microorganisms (A) that contain at least one heterologous DNA fragment (I) containing a selected gene (II) that can be expressed in the digestive tract and have survival rates over 25%, particularly 90-95%, when leaving the ileum after 6 hours of digestion and over 2%, preferably 8%, after 12 hours fermentation in the colon. Independent claims are also included for the following: (1) method for studying, monitoring and/or selecting (A), using an artificial digestive system; (2) (A) produced by method (1); and (3) combined product containing (A) and an active substance, or its precursor, for simultaneous, separate or staged use.

Description

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The present invention relates to the development of genetically modified microorganisms, including genetically modified yeasts, which can survive in a digestive environment and effectively express one or more genes of interest. The dosage form and the mode of administration of these microorganisms are also provided. In addition, the invention relates to the use of artificial digestive systems simulating the gastrointestinal functions of humans for the study, control and selection of the aforementioned microorganisms. The invention also relates to direct applications of in situ bioconversion, in particular in the development of preventive or curative treatments for pathologies, particularly diseases related to xenobiotic metabolism.

Living beings are constantly in contact with xenobiotics, substances that are foreign to the normal metabolic pathways of the cell (drugs, pesticides, pollutants, food additives, natural molecules of plants ...). These compounds, which are often too hydrophobic to be eliminated directly by the kidneys or degraded by bile, accumulate in the lipids of the body. The enzymes of xenobiotic metabolism (or EMX) are responsible for facilitating their elimination by making them more hydrophilic.

These enzymes are classified in three categories: - phase I enzymes (functionalization phase): the more hydrophilic xenobiotic by introduction of polar groups (usually by oxidation reactions). These are mainly the cytochrome P450 monooxygenases, hemoproteins with a characteristic absorption maximum around 450 nm, when they are reduced and bound to carbon monoxide (Omura, 1964, 1993). In mammals, P450s are predominantly in the smooth endoplasmic reticulum membrane, mainly in the liver, but also in the lungs and intestine, which play a role in controlling the entry of many xenobiotics into the body. 'organization.

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Each P450 is part of a multienzyme complex where it is associated with two flavoprotein electron transport systems, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase.

P450s operate according to a catalytic cycle in which the iron passes through different degrees of oxidation, which leads to the incorporation of an oxygen atom in the substrate R according to the following reaction (Guengerich, 1993): RH + O2 + 2e- + 2H + # ROH + H20 P450 possess original catalytic properties since, unlike the majority of enzymes, a given P450 can act on many substrates with very different structures and, conversely, the same substrate can be recognized by different P450s (Coon et al., 1992). Endogenous factors (species, strain ...), physiological factors (age, sex ...), but also exogenous factors can modulate the activity of P450 (Guengerich, 1993), by acting on different levels: gene expression, stabilization of the protein, modulation of the activity.

 phase II enzymes (conjugation phase): metabolites from phase 1 that are even more hydrophilic (if they are not excreted directly), by conjugation with various endogenous molecules (glutathione, glucuronide, etc.). These enzymes are present in almost all cells of the body, the main ones being glutathione S-transferase (GST), N-acetyl transferase (NAT), quinone reductase (QR), aldehyde dehydrogenase (ALDH), uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase, epoxide hydrolase, and O-acetylase.

- phase III enzymes (evacuation phase): responsible for eliminating metabolites from detoxification reactions (ABC transporters, especially multidrug resistant MDR pumps).

Overall, the metabolism of xenobiotics is considered a detoxification pathway. However, phase 1 enzymes may give rise to metabolites that are more reactive than the initial molecule, that can bind to proteins (causing necrosis or autoimmune diseases), nucleic acids (responsible for mutagenesis or carcinogenesis) or lipids (peroxidation

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 lipid). This is particularly the case when phase II enzymes are absent or activity too low.

The activity of EMX is genetically determined and highly variable according to individuals (genetic polymorphism). Molecular analysis of correlations between major genes involved in detoxification and biotransformation processes and pathologies is currently the subject of much research. Indeed, abnormalities in the detoxification and biotransformation processes of exogenous compounds have clearly been identified in patients suffering from so-called multifactorial diseases (diseases combining genetic predisposition and the role of environmental factors), which are widespread in the world today, such as that different cancers, various digestive pathologies, endometriosis, chronic bronchitis ...

For example, the risk of lung cancer induced by polycyclic aromatic hydrocarbons is increased by about 40-fold when the individual is deficient in GSTM1 (GSTM1 0/0 phenotype) and has a P450 1A1 with high enzymatic activity (Raunio et al. ., 1995). Furthermore, according to epidemiological studies, the populations at greatest risk of developing colon cancer are those with the fast metabolizing phenotype for NAT2 and P4501A2 and who are exposed to high levels of heterocyclic amines in their body. diet (Lang et al., 1994). Regarding breast cancer, correlations have also been established. Women with a highly inducible P450 1A1 form are at increased risk for breast cancer. Similarly, the risk of breast cancer is increased (x4) in postmenopausal women, smoking moderately (presence of aromatic amines) and having a slow phenotype for NAT2. The group at risk for bladder cancer is represented by individuals deficient in GSTM1 and NAT2 (Brockmoller et al., 1996). A positive correlation has been established between endometriosis and GSTM-deficient women 1, as well as the slow metabolizing phenotype for NAT2 (Baranova et al., 1999).

Diet is one of the main sources of xenobiotics involved in

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 multifactorial diseases, hence the interest of ensuring the detoxication of these xenobiotics before they exert negative effects on the target organs.

Thus, the present invention relates to the development of a living medicament, genetically modified microorganisms, and more particularly yeasts, expressing in the digestive environment one or more gene (s) of interest (s) for correcting dysfunctions. metabolism of xenobiotics, such as those mentioned above. This or these genes of interest will be chosen after determining the correlations between genetic heritage and the presence of pathology and after evaluating the prevalence of this pathology.

In addition to the main detoxification application, two other objects of the present invention are described below, by way of example.

The first is to express in the digestive environment by said microorganisms a gene of interest encoding an enzyme for in situ activation of pre-substance active substance. The administration of such a living medicament for controlling the bioconversion of pre-substance into active substance is particularly advantageous in the case where the active substance has undesirable side effects and / or is toxic from a certain dose. Among these applications, mention may be made of the transformation of provitamines into vitamins.

The second is to produce in the digestive environment by these microorganisms an active peptide or polypeptide, in particular having antigenic properties. The production of such peptides, at different points in the digestive tract, leads to the development of live vaccines which make it possible to circumvent a certain number of difficulties encountered in the conventional methods of administration belonging to the state of the art, such as the problems degradation.

Fahl et al (WO 99/27953) constructed E.coli strains expressing mammalian enzymes (P450 1A1, NADPH-cytochrome P450 reductase and GST) whose genes were inserted into a plasmid. These model constructions are carried out in order to develop chemoprotective bacterial strains, in particular

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 Lactobacilli, expressing mammalian enzymes involved in the detoxification of procarcinogens and administered in the digestive environment to supplement the detoxification system of gastrointestinal cells.

Fahl et al. do not specify the survival time of Lactobacilli genetically modified in a digestive environment and whether they can effectively express a gene of interest.

To answer these questions, we have set up artificial digestive systems that make it possible to study, adapt, control and select micro-organisms that survive at least for a minimum time and that can express effectively in situ ( that is to say in the digestive environment) one or more heterologous genes of interest. Of course, the survival time depends on the microorganisms in question and the digestive environment, but it can be defined in the context of the invention this parameter being greater than 6h in the small bowel stomach and 48H in the colon, that is to say the average duration of transit in these digestive compartments in humans.

Among the preferred microorganisms of the invention, mention may be made of yeasts. Indeed, in the same way as the bacteria referred to in the aforementioned patent application, the yeasts are organisms present in the animal and / or human diet and well-known hosts of the digestive environment. Some of them are GRAS or recognized as safe organisms and have been used for some years as probiotics.

Their ease of production explains their large-scale use in the food industry, for example in the processes of making beer and bread or as an additive in animal and human nutrition. S cerevisiae is the best known and the most commercially exploited. Today, S cerevisiae and S boulardii are also used in the pharmaceutical industry as drugs (carbolevure and ultralevure # respectively), mainly for the treatment of disorders secondary to antibiotic therapy such as diarrhea, colitis or candidiasis (Ligny G, 1975 ). Many studies conducted in animals and humans highlight both the safety and the positive impact of these yeasts on the digestive environment (antagonism towards Candidas for example).

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Compared with bacteria, the use of yeasts for living medicines has considerable advantages: - Unlike bacteria, yeasts are eukaryotic organisms.

The genomic integration of heterologous fragments is easily feasible (highly efficient homologous recombination) and results in a more stable construction than that obtained by plasmid insertion, which in particular limits the potential transfers of the gene of interest to the human endogenous flora.

In addition, yeasts have a subcellular organization very close to that of higher eukaryotes and have intracellular transport mechanisms and post-translational processing essential for the functional and compartmentalized expression of many heterologous genes. For example, they have been shown to possess the criteria necessary for the synthesis of functional human P450s since a protein addressing system ("Signal Recognition Particle" or SRP) allows the translocation of synthesized P450 to the endoplasmic reticulum, resulting in correct P450 intracellular localization and folding (Urban et al., 1994a, Pompon et al., 1997). Yeast also has a lipid membrane environment that allows the stability of P450 and electron transfer proteins (NADPH-cytochrome P450 reductase and cytochrome b5) in the endoplasmic reticulum membrane, necessary for the enzymatic activity of P450.

- Yeasts, well characterized at the molecular level (in particular S cerevisiae, fully sequenced since 1996-Goffeau et al.-), constitute a basic tool for genetic engineering and bioconversions in vitro under fermentation conditions not only in the laboratory but especially on a large scale (industrial production).

Some yeasts such as S cerevisiae have the capacity to live aerobically and anaerobically, which makes it possible to consider the use of such yeasts at any level of the digestive tract.

- Other advantages concerning the construction of recombinant yeasts are also to be emphasized. Indeed, the yeasts are selected by auxotrophy and not on the basis of resistance to an antibiotic, which avoids being confronted with

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 problems related to the administration to humans of genetically modified organisms having an antibiotic resistance gene. In addition, the genomic insertion of the gene of interest is facilitated by the recombination property of certain yeasts, including S cerevisiae (Bellamine, 1996).

Strains of S. cerevisiae have been developed allowing the expression of three major types of genes involved in the different phases of xenobiotic metabolism: human cytochrome P450 genes (phase I), "phase environment" enzyme genes I "(NADPH-cytochrome P450 reductase and cytochrome b5 for example) and phase II genes. Such strains have been described by Pompon et al. (US 5,635,369 and EP 595,948).

In the context of the present invention, S. cerevisiae strains have been used as study models. They express on the one hand the human P450 1A1 and the yeast or human NADPH-cytochrome P450 reductase (strains W (R) pHlAl and W (hR) pH1Al, respectively), on the other hand the plant P450 73A1 and the Yeast NADPHcytochrome P450 reductase (strain W (R) pP73Al).

P450 1A1 exists in the majority of animal species, including humans.

aromatiques halogénés tels que la 2,3,7,8-tétrachlorodibenzo -dioxine (TCDD) ou par des flavonoïdes comme la (3-naphtoflavone. The human form has been isolated by Jaiswal et al. (1985). It is involved in the metabolism of polycyclic aromatic hydrocarbons, particularly in the case of benzo (a) pyrene, a compound found in particular in cigarette smoke and barbecue products (Sims et al., 1974, Gautier et al., 1996a). It is difficult to detect in the liver without the action of an inducer but exists in many extrahepatic tissues in the constitutive state. It is strongly inducible by polycyclic aromatic compounds such as 3-methylcholanthrene (3-MC), by compounds

halogenated aromatics such as 2,3,7,8-tetrachlorodibenzo-dioxin (TCDD) or flavonoids such as (3-naphtoflavone.

P450 73A1 (cinnamate 4-hydroxylase or CA4H activity) catalyzes the first stage of the phenylpropanoids pathway in higher plants by transforming trans-cinnamic acid into p-coumaric acid. This path is the beginning of synthesis

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 flavonoids (antioxidants).

The survival of model yeasts, the maintenance of their activity after passage into artificial digestive systems and their activity in situ have been demonstrated in the context of the present invention. Such survival abilities and enzyme activity prove to be very important if one wants to use living microorganisms as a drug.

In this respect, the method for evaluating the fate of drug microorganisms in the human digestive environment is also an object of the present invention.

As mentioned above, one of the main problems encountered in the development of a living drug lies in its selection, in the evaluation of its survival, its activity and its control, in a digestive environment. To circumvent these difficulties, the present invention proposes the use of artificial digestive systems reproducing in the most reliable and dynamic manner the human digestive environment. Technically advantageous, these systems allow the control of the main parameters of digestion and ensure the reproducibility of experimental conditions, factors that can not or very difficult to control in laboratory animals. In addition, these systems make it possible to take samples at any level of the digestive tract and to overcome all the ethical constraints related to animal or human experiments (potentially toxic molecules). Such systems also provide the opportunity to evaluate the impact of genetically modified micro-organisms on human endogenous flora (implanted in the artificial digestive system).

Thus, the artificial digestive systems make it possible to carry out a selection of genetically modified microorganisms, in particular genetically modified yeasts, which have not only adapted to the digestive environment but which retain a good capacity to express a heterologous gene of interest.

Any artificial digestive system incorporating the essential characteristics of the human digestive system can be used within the scope of the invention. Various in vitro systems

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 very simple (individualized digestive compartments) have been described. Their use is however limited by the fragmentation of the digestive compartments and the lack of dynamism. It is therefore advantageous to use the complete artificial digestive system described in WO 94/09895.

Another advantage of the present invention stems from the specific formulation of the living drug so as to protect the microorganisms and to specifically target an action in a particular part of the digestive system.

The microorganisms, and more particularly the yeast, to be active in the gastrointestinal tract, the route of administration is the oral or rectal route. The recombinant microorganisms must be administered in a galenic form that allows them to be transported to their place of action, to keep them in an active state and to protect them from the external environment. The artifices or galenical processes used to achieve these objectives are different according to the sensitivity of microorganisms to the digestive environment and according to the places where they will have to carry out their activity (level of the gastrointestinal tract). Among the galenical methods used, mention may be made of: the lyophilization of the microorganisms and their nutrient medium, the encapsulation of the microorganisms or the coating of the galenic system enclosing them with the aid of polymers that are sensitive to pH or that possess mucoadhesive properties, mixing with polymers having muco-adhesive properties, compression of microorganisms in the presence of excipients having muco-adhesive properties, incorporation into hydrophilic or lipophilic gels.

The resulting galenic systems are monolithic monolayers such as monolayer or multilayer tablets, gelatin capsule type (hard or soft) or multiparticulate microgranules or microcapsules. These forms allow normal or delayed release of microorganisms within the digestive tract.

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Thus, in a first aspect, the present invention relates to microorganisms comprising at least one heterologous DNA fragment comprising one or more genes of interest capable of being expressed in the digestive environment.

Advantageously, said microorganisms are also characterized in that they can survive in the digestive environment for at least 6h, 10h, 12h or 24h in the stomach and small intestine and / or at least 12h, 24h, 60h or 72h in the colon.

Indeed, in the artificial digestive systems described below, microorganisms are obtained whose survival rate is greater than 25%, 50%, 75%, preferably 80% at the outlet of the ileum after 6 hours of digestion and is greater than 2%, 4%, preferably 8% after 12 hours of fermentation in the colon.

These measurements can easily be made by enumeration from readily digestible samples in artificial digestive systems, at any time during digestion or fermentation.

In the context of the invention, yeasts are selected, preferably yeasts selected from Saccharomyces cerevisiae, S boulardii, S uvarum, Schizosaccharomyces pombe and Klevuromyces lactis. Bacteria such as lactobacilli described in Fahl et al (WO 99/27953) can also be selected.

Genes of interest Genes of interest are genes coding for peptides of interest for vaccination, peptides of hormonal interest (such as insulin), peptides of therapeutic interest (such as anti-proteases, coagulation factors). peptides with an antibiotic effect, peptides capable of trapping or complexing molecules, enzymes or any peptides having bioconversion or biosynthesis activities, in particular detoxification enzymes.

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In the sense of the invention, the terms polypeptides, peptides or proteins may be used interchangeably from each other and in no case constitute a limitation to a given size.

The genes of interest will preferably be selected from the enzymes of the metabolism of xenobiotics, namely phase 1 enzymes (P450 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 73A1), phase 1 environment enzymes (NADPH-cytochrome P450 reductase and cytochrome b5), phase II enzymes such as glutathione S-transferase (GST), N-acetyl transferase (NAT), quinone reductase (QR), aldehyde dehydrogenase (ALDH), uridine diphosphoglucuronosyl transferase (UGT), sulfotransferase, epoxide hydrolase and O-acetylase, and phase III enzymes (ABC transporter pumps, particularly multidrug resistant pumps). MDR).

Glutathione S-transferase is a gene superfamily composed of 4 main classes: alpha (GSTZ), mu (GSTM), pi (GSTP) and teta (GSTT).

These enzymes play an essential role in cell resistance against lipid peroxidation, DNA damage and protein alkylation caused by electrophilic chemical species. They catalyze the binding of many electrophilic xenobiotics to glutathione and are involved in the detoxification of oxygen radicals. The different classes of GST are present in large quantities in many tissues, with a certain tissue specificity depending on the class considered.

The genes encoding these different enzymes have been located on human chromosomes. Various polymorphisms have been demonstrated, in particular concerning the gene coding for GSTM 1. Indeed, three different alleles of GSTM1 have been identified: the GSTM 1 A and GSTM 1 B alleles encode enzymes of similar activity while the GSTM10 allele codes for a non-functional enzyme. In most of the populations studied globally, 35% to 50% of subjects are homozygous for the GSTM10 allele and therefore lack the corresponding enzymatic activity. Due to this failure of their detoxification system, individuals of genotype GSTM10 / 0 are considered at-risk individuals when exposed to high levels of carcinogens and chemicals

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 toxic. Indeed, the GSTM1 deficiency is associated with an increased risk of cancer (mainly lung and bladder cancers) and some chronic diseases (endometriosis, chronic bronchitis ...).

Two N-acetyltransferases (NAT1 and NAT2) have been identified, catalyzing N-acetylation reactions of arylamines, as well as other detoxification reactions. They are also involved in the metabolism of drugs such as sulphonamides and in drug interactions. The variations in NAT activity correspond to their ability to transfer Acetyl Coenzyme A acetate to the substrate.

Only the NAT2 gene has a polymorphism allowing differentiation between slow acetylator (SA) and fast acetylator (RA). Available studies suggest that slow acetylation is more of a risk factor than rapid acetylation. Indeed, the SA phenotype is considered a risk factor for bladder cancer. In addition, menopausal women with the SA phenotype are 4 times more likely to develop breast cancer than women with the RA phenotype.

Construction of recombinant microorganisms: for example yeasts S. cerevisiae strains have been developed allowing the expression of three major types of genes involved in the different phases of xenobiotic metabolism. In this context, a series of expression cassettes for mammalian genes (human, mouse, rabbit) or plants (Jerusalem artichoke, for example) has been produced, cassettes carried by autonomous multi-copies vectors or integrated into the DNA. chromosomal yeast. For example, the human P450 expressed in yeasts are the 1A1, 1A2, 2B6,2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 19A isoenzymes.

In parallel, coexpressions of phase 1 genes, phase I (NADPH-cytochrome P450 reductase and cytochrome b5 for example) and phase II genes have been developed. The effectiveness of these constructions has been demonstrated. For example, the three stages of biotransformation of benzo (a) pyrene P450 1A1, NADPH-cytochrome P450 reductase and epoxide hydrolase were reproduced

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 in the yeast S cerevisiae: the analysis of the products of incubation of benzo (a) pyrene with these recombinant yeasts shows the formation of all the expected metabolites (Gautier et al., 1996b).

Such systems can be used as a model for the study of certain aspects of human metabolism, as an aid to the analysis of the toxicity of pollutants and especially as a bioconversion tool.

It is possible, after designing selection markers and viability testing of quite original types, to demonstrate and control the survival and, if necessary, death and elimination of functional recombinant yeasts in the human digestive environment.

It is possible to identify and to create by molecular engineering control structures of heterologous genes (promoter sequences in particular), allowing an adapted and adaptable activity in the human digestive environment.

For purposes of expression of the gene of interest, constitutive or inducible promoters, strong promoters, promoters that are active specifically in a segment of the digestive tract under aerobic, semi-anaerobic or anaerobic conditions may be used. promoters regulable by a molecule introduced into the intestinal medium. It is possible to develop a genetic engineering technology that meets the essential criteria for in vivo use in humans, in particular the absence of mobilizable vectors, the absence of markers of antibiotic resistance, a conditional genetic stability of functions heterologous allowing their destruction by an external chemical factor if necessary (interruption of the treatment for example).

The heterologous DNA fragment may further have a genetic marker for selection or counter selection or a genetic bar code.

Study and selection of recombinant microorganisms using artificial digestive systems In a second aspect, the invention relates to a method for studying, controlling, adapting and / or selecting the recombinant microorganisms described below. over at

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 means of an artificial digestive system.

By artificial digestive system is meant any device incorporating the essential characteristics of the human digestive system.

The artificial digestion system can be defined as a reaction device described in WO 94/09895.

This system is the most comprehensive model in the world today. Indeed, it has all the compartments of the digestive system (stomach, duodenum, jejunum, ileum and colon, figures 1 and 2 below) and reproduces as faithfully as possible the digestive environment of the man or the monogastric animal. It takes into account the essential parameters of digestion such as peristaltic movements, gastric and intestinal pH variations, gastric emptying and intestinal transit time, gastric, biliary and pancreatic secretions, the absorption of digestion and fermentation, water absorption, faecal microflora (Minekus et al., 1995 and 1999). Computer control of the system allows continuous control of the parameters. This system allows to mimic different situations, both physiological and pathological. It has many advantages in terms of reproducibility, control of experimental conditions (difficult to control in laboratory animals), standardization, reliability, speed, ability to collect samples at any level of the digestive tract, while limiting large numbers of technical constraints and problems of acceptability (impossibility of carrying out experiments with potentially toxic molecules in humans).

Concretely, each compartment is formed of glass units containing flexible internal walls. A passage of water between the flexible walls and those of glass makes it possible to maintain a constant temperature of 37 C and to mimic the peristaltic movements (thanks to variations of pressure of the water). The different compartments are interconnected by peristaltic valves, whose opening is controlled by computer (sending or not nitrogen).

The passage of the chyme from one compartment to another is controlled by the amount of

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 food present in each compartment (presence of level sensors).

An exponential formula is used to model gastric and ileal emptying (Minekus et al., 1995): f = 1-2- (t / t1 / 2) B; where f represents the delivered meal fraction, t1 / 2 the meal release half-time, t the meal release time, and B a parameter describing the shape of the curve).

The volume of the digestive contents is recorded continuously by a pressure sensor and kept constant by water absorption through dialysis fibers by means of a pump.

Stomach (TIMI) The amount of food to be introduced into the artificial stomach and the duration of digestion (Minekus et al., 1995) are chosen. Pepsin is delivered continuously through a pump. The pH is continuously readjusted with HCl or water to follow a predetermined curve established according to in vivo data.

The small intestine (TIMI) The artificial small intestine is programmed to simulate a transit time as close as possible to that observed in vivo. The pH is maintained at the most physiological possible values by NaHCO3 (6.5 in the duodenum, 6.8 in the jejumum and 7.2 in the ileum). In the duodenum, pumps provide pancreatic enzymes (mixture of pre-enzymes that will be activated by trypsin) and bile acids. The absorption of water and digestion products is achieved through a dialysis system operating with hollow fiber units, connected to the jejunum and the ileum.

The colon (TIM2) In its basic form, the artificial colon consists of a loop where a previously installed faecal flora cyclically circulates. It was verified that the composition and the fermentative activity of this faecal microflora, stool from volunteer donors and implanted in the digestive system, were well representative of those of the human colonic flora and that this flora was stable and

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 over time (Minekus et al., 1999).

A stream of nitrogen keeps the system anaerobic, condition controlled by an indicator (resazurin solution). The nutritional medium is stored in a glass unit maintained at 4 C where a nitrogen influx allows its permanent mixing in anaerobic. A set of dialysis fibers crossing the entire colon can mimic the absorption of water and nutrients. The pH is kept constant and adjusted to 6.5 by NaOH solution (instead of bicarbonate in vivo). Total gas production (such as H2, CO2 and CH4) can be determined by measuring the amount of water displaced in a Mariotte bottle. The different gases, as well as the volatile fatty acids produced (mainly acetate, butyrate and propionate), can be analyzed qualitatively and quantitatively by gas chromatography.

In a more elaborate form, the artificial colon consists of three subunits simulating the three parts, proximal, transverse and distal, where different fermentative activities (associated with different pHs) occur.

Of course, all or part of the aforementioned artificial digestive systems can be implemented within the scope of the invention.

Thus, the invention relates to a method for studying and controlling microorganisms comprising the steps of: a) adding the microorganisms according to the invention in a liquid nutrient medium, b) introducing said medium to the the entry of a system as defined above, c) incubating and counting the recombinant microorganisms and / or testing the activity of the gene (s) of interest after passage into the digestive systems or in situ. For example, this step may include a PCR test.

In another embodiment, the method may comprise the steps of: a) adding the microorganisms according to the invention in a liquid nutrient medium, b) introducing said medium into the inlet of a system as defined herein. above, c) incubate and recover surviving microorganisms after passage through TIM 1

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 and / or TIM2; possibly repeat steps a) to c) until a homogeneous population of microorganisms adapted to the digestive environment and capable of effectively expressing the gene (s) of interest is obtained.

The invention also relates to microorganisms that can be obtained by the process mentioned above. These microorganisms may be characterized in that their survival rate is greater than 25%, 50%, 75%, preferably 80% at the outlet of the ileum after 6 hours of digestion and is greater than 2%, 4%, preferably 8% after 12 hours of fermentation in the colon. Preferably, it is yeasts.

The survival of microorganisms can be assessed by counting. The presence of the gene (s) of interest can be demonstrated by molecular techniques (for example Polymerase Chain Reaction). The expression of the gene (s) of interest can be estimated by biochemical techniques such as mono or bi-dimensional electrophoresis or Western Blot. The activity of the expression products of the gene (s) of interest is evaluated by specific techniques (for example, enzymatic activity assay).

Method of administration of microorganisms Know-how in the field of galenics and biopharmacy to optimize the administration of active ingredients orally have been developed. The effectiveness of a drug is conditioned by its bioavailability, that is to say the proportion of active ingredient arriving at the level of the general circulation, and according to which kinetics. One of the aims of the galenic formulation is therefore to produce an optimized form that is best adapted to the treatment of a particular disease or syndrome, ie a form presenting easy administration for the patient and good availability. .

For the classical active ingredients (origin and chemical nature defined), the oral route is one of the preferred routes of contribution, by its convenient administration and its

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 low cost. The active ingredients thus administered may be absorbed throughout the gastrointestinal tract. They will eventually undergo different levels of biodegradation under the action of enzymes, digestive juices and pH variations.

The fraction absorbed in the digestive tract may reach the site of action after passage through the liver and possible metabolism.

In contrast, in the case of microorganisms, they will not be absorbed by the intestinal mucosa but will remain in the tract for a longer or shorter time in order to exert their action at the level of the defined target. The galenic form containing the microorganisms is a form allowing to bring them intact at the level of the target and to maintain their integrity at this level for a defined time more or less long.

Under these conditions, the techniques used with conventional active principles to ensure sustained release apply to microorganisms after adaptation of the technology and operating parameters.

All further details concerning these dosage forms are available in EP 904021995 (galenical forms improving bioavailability), EP0542824 (muco-adhesive forms) and FR 9806958 (oily gel and bigels).

These different techniques or processes, as well as any other method of the state of the art of coating and encapsulation, can be applied to the microorganisms of the invention.

For substances that are sensitive to atmospheric humidity or those sensitive to oxidation by oxygen in the ambient air, protection is necessary for the preparation of dosage forms (capsules, tablets) in order to guarantee their preservation and maintain their activity. therapeutic. The coating allows direct deposition of protective agent (resin, polymer) to protect the active ingredient. In the case of microorganisms, in particular yeasts, according to the invention, the coating is intended to isolate them from the hostile external environment in order to protect them from the aggressions of

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 enzymes, pH and juices present at the digestive level. The result of the coating is the obtaining of a sphere or microsphere whose diameter is directly proportional to the amount of coating product used and the size of the product to be coated at the origin. The operating parameters, the technical equipment and the adjuvants used to make this coating are important factors for obtaining microspheres having defined characteristics. The coating is either a pH sensitive polymer solubilizing at a given pH of the gastrointestinal tract, a polymer including the nutrients necessary for the survival of microorganisms, or a mucoadhesive product facilitating the mucoadhesion of microorganisms in the body. level of the gastrointestinal tract, a combination of these different compounds. The result obtained consists of microspheres which are then packaged in hard or soft capsules.

Thus, another aspect of the invention relates to a pharmaceutical composition comprising the microorganisms defined above advantageously in a dosage form suitable for oral or rectal administration so as to route them intact at the target site of the tract. digestive and maintain their integrity at this level for a given time.

In this pharmaceutical composition, the microorganisms, in particular yeasts, may be protected by an associated composition or covered by a coating and / or a membrane of the resin and / or polymer type forming spheres, microspheres, granules or microgranules. .

Preferably, the composition is in the form of capsules, tablets, or dragees.

The invention also relates to a combination product comprising the microorganisms described above and an active substance or a precursor of an active substance for simultaneous use, separated or spread over time.

This product is preferably characterized in that said microorganisms interact with the active substance or the precursor. The active substance is intended to promote the action of recombinant microorganisms (for example, it may be a substance

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 promoting the survival, growth or activity of microorganisms) or, conversely, the microorganisms act on the active substance or on the precursor.

Conclusion Another aspect of the invention relates to the use of microorganisms according to the invention for the preparation of a medicament such as detoxification agents, metabolic correction agents, agents capable of modifying the intestinal flora. agents capable of increasing the bioavailability of an active substance or its precursor and for the preparation of a drug capable of providing in situ active peptides, peptides of vaccine interest, peptides of hormonal interest, peptides therapeutic interest, peptides with antibiotic effect or peptides capable of trapping or complexing molecules.

Figures 1 and 2: schematic diagrams of artificial digestive systems.

Figure 1 (Artificial stomach and small intestine or TIM 1) Figure 2 (Artificial colon or TIM 2) Figure 3: Genomic and plasmid constructs for the expression of the human P450 1A1 gene and human NADPH-cytochrome P450 reductase (A) or yeast (B) and for expression of the P450 73A1 gene of Jerusalem artichoke and yeast NADPH-cytochrome P450 reductase (C).

A- The strain obtained is called W (hR) pHIAl.

B- The strain obtained is called W (R) pH1A1.

C- The strain obtained is called W (R) pP73A1.

URA 3: wild orotate decarboxylase; ADE 2: wild-type aminoimidazole ribonucleotide carboxylase gene; ura 3: mutated orotate decarboxylase; Y red: yeast P450 reductase; H red: human P450 reductase; PGK phosphoglycerate kinase; Y red ORF: coding sequence of P450 reductase

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 yeast; H red ORF: coding sequence of human P450 reductase, H 1A1 ORF: coding sequence of human P450 1A1; P 73A1 ORF: coding sequence of P450 73A1 of Jerusalem artichoke; pro: promoter; ter: terminator.

FIG. 4: release kinetics of the recombinant yeast W (hR) pHIA1 and W (R) pHI11 at the outlet of the ileum.

The results obtained during three digests in TIM1 are presented digestion by digestion (A) and averaged (B).

The results are expressed as cumulative levels of surviving yeasts recovered at the outlet of the ileum relative to the number of yeasts ingested. For each sample (t = 120, t = 240 and t = 360 min) corresponding to a collection interval of the digestates (0-120 min, 120-240 min and 240-360 min), the total number of yeast is listed and expressed as a percentage of the total number of yeasts ingested. These percentages are then cumulated over time.

The dilution factor due to the volume of digestive secretions is taken into account in the calculations.

Figure 5: Monitoring recombinant yeast W (hR) pHlA1 and W (R) pHI1l in the artificial colon.

Various samples were taken in order to be able to carry out yeast monitoring in the TIM 2: at t = 0, the number of cells per ml of suspension of recombinant yeasts is listed before introduction into the colon, at t = Ofec: the number of endogenous yeast cells per ml of colonic content is enumerated, before colon seeding, at t = 1, t = 12, t = 24, t = 36, t = 48 and t = 60h: the number is enumerated yeast cells (recombinant and endogenous) per ml colonic content taken from the colon.

The results obtained are expressed in number of cells per ml (logarithmic scale) taking into account the dilution factor due to the volume of the colic content.

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FIG. 6: results of the PCR carried out on the W (R) pH1A1 and W (hR) pH1A1 strains before passing through the digestive systems (A), at the exit of the small intestine (B) and the colon (C) A: before passing through the digestive systems (t = 0min) B: leaving the small intestine (t = 120 min) C: leaving the colon (t = 12h)
Primers (see Figure 3) 6 + 7 '3 + 4 1 + 7 1 + 8 5 + 2' 3 + 4 1 + 2 1 + 8 3 + 4 3 + 4
Size of fragments (bp) 593 650 351 459 461 650 338 459 0 650
W (R) strain pH1A1 W (hR) pH1A1 T <0 T> 0 T = Control Figure 7: conversion kinetics of trans-cinnamic acid into pcoumaric acid in the different digestive compartments of TIM1, under the action of W (R) pP73Al.

7A: stomach; 7B: duodenum; 7C: jejunum; 7D: ileum.

FIG. 7 represents the ratio of the number of moles of p-coumaric acid to the number of moles of trans-cinnamic acid present in the various compartments of TIM 1 (stomach, duodenum, jejunum and ileum) during digestion. .

The sampling times are given in Example 5 below.

Example 1: The yeast Saccharomyces cerevisiae as an expression system of human P450.

S. cerevisiae was the first system used for the expression of a rat P450 (Oeda et al., 1985). Since then, it has been widely used as a heterologous expression system in many laboratories. In particular, the team of Dr. Denis Pompon chose it as a study model for the expression of human P450 (isoenzymes 1A1, 1 A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3 A4, 3A5, 19A).

Use yeast as a host for the heterologous expression of P450 exhibits

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 many advantages. Indeed, yeasts possess the criteria necessary for the synthesis of functional human P450s (Urban et al., 1994a, Pompon et al., 1997). They present the intracellular architecture and the majority of posttranslational modifications of eukaryotic cells, as well as a protein recognition system ("Signal Recognition Particle" or SRP) allowing the translocation of synthesized P450 to the endoplasmic reticulum, which leads to correct intracellular localization and folding of P450. Yeast also has a lipid membrane environment that allows the stability of P450 and electron transfer proteins (NADPH-cytochrome P450 reductase and cytochrome b5) in the endoplasmic reticulum membrane, necessary for the enzymatic activity of P450.

The introduction into yeasts of expression cassettes can be carried out in two ways: on plasmid or by genomic integration.

In addition, the yeasts make it possible to coexpress stably several different complementary cDNAs (cDNAs) and the techniques used are low cost and without risk.

The S. cerevisiae yeast allows the characterization of human monooxygenase activities (Urban et al., 1994a, Gautier, 1994), namely: the study of substrate specificity in a context free of contaminating monooxygenase activity (S cerevisiae) contains weakly expressed endogenous P450 whose activity is not likely to interfere with that of human P450), - the study of genetic diversity and enzymatic polymorphism (possibility of expressing scarce P450 forms), - study of the structure / function relationship by site-directed mutagenesis and by the fabrication of fusion proteins (search for amino acids involved in a particular aspect of human P450 function).

Despite the advantages previously enumerated, the use of yeasts as a heterologous expression system (Urban et al., 1994a, Pompon et al., 1996) has some limitations:

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 - the subcellular fractions are difficult to prepare because of the very resistant wall of the yeasts, - the level of the endogenous NADPH-cytochrome P450 reductase is weak, which limits the activity of the expressed P450, - the enzymes NADPH-cytochrome P450 reductases yeast and human have low sequence similarity (33%), which decreases the coupling efficiency between human P450 and yeast NADPH-cytochrome P450 reductase, - human P450 production (between 50 and 500) pmol of P450 per mg of microsomal protein) is lower than in the bacterium E coli (of the order of nmol / mg) but this disadvantage is largely offset by the fact that, unlike the bacterium, the yeasts produce functional P450.

In order to better mitigate some of these limits (Urban et al., 1994a, Pompon et al., 1995), different expression systems have been developed: - First-generation systems: the expression of human P450.

An expression cassette, inserted in a plasmid, allows the production of a human P450. To obtain a sufficient level of expression, the 5 'and 3' non-coding sequences of the cDNA were deleted before insertion into the yeasts. In addition, the strong promoter GAL10-CYC1 (totally repressed by glucose but strongly inducible by galactose) was introduced in order to separate the exponential growth phase, required to constitute the biomass, from the expression phase.

 - Second generation systems: the specific activity of human P450.

Several types of genomic modifications have been performed to increase the specific activity of P450 by 10 to 100 times that obtained with first-generation systems: - the construction of a fusion protein between human P450 and yeast NADPHcytochrome P450 reductase the overexpression of yeast NADPH-cytochrome P450 reductase, placed under the control of the GAL10-CYC1 promoter,

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 the coexpression of human cytochrome b5 and human P450.

Coexpression of phase II enzymes (such as epoxide hydrolase) and human P450 also allowed for the simulation of metabolic couplings between phase 1 and phase II enzymes (Gautier et al., 1993).

- Third generation systems: "humanisation" of the redox environment.

At the genome level, the yeast NADPH-cytochrome P450 reductase was replaced by a human reductase, which caused a sharp increase in the specific activity of human P450 x10 to 500 compared to first-generation systems.

Example 2: Obtaining recombinant yeast strains W (hR) pHlA1, W (R) pH1A1 and W (R) pP73A1 The strain of S cerevisiae W (hR) pHI1 (FIG. 3A) is derived from the strain W (hR). two strains W (R) pHIA1 (FIG. 3B) and W (R) pP73A1 (FIG. 3C) are derived from the strain W (R).

 The construction of the W (R) W (hR) strains was described by Truan et al. (1993) and Urban et al. (1993).

Genomic modifications have enabled the construction of the W (R) W (hR) strains from laboratory strain W3031 B (sexual sign MAT a), which possesses mutations on certain genes (leu 2, his 3, trp 1, ura 3, ade 2), thus allowing its selection by auxotrophy.

 W (R) strain: open reading phase of yeast NADPH-cytochrome P450 reductase (2072 bp) was overexpressed by insertion of the strong promoter GAL10-CYC1 (480 bp) in place of the promoter of NADPH-cytochrome P450 reductase yeast.

 The terminator of the yeast NADPH-cytochrome P450 reductase gene has been conserved.

Strain W (hR): open reading phase of the human NADPH-cytochrome P450 reductase (2034 bp) was inserted into the genome of the yeast in place of that of the
NADPH-yeast cytochrome P450 reductase and placed under the control of the sponsor

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 strong GAL10-CYC1. The terminator of the yeast NADPH-cytochrome P450 reductase gene has been conserved.

The two strains W (hR) pH1A1 and W (R) pHI11 respectively result from the transformation of W (hR) W (R) with the plasmid pi A1.

Plasmid pi A1 (see FIG. 3) results from the insertion into the plasmid pV60 of the coding sequence for human P450 1A1 (1539 bp), placed between the strong promoter GAL10-CYC1 and the terminator of the phosphoglycerate kinase gene ( PGK). It contains the gene encoding ss-lactamase that confers ampicillin resistance in E. coli.

This plasmid can be maintained in yeasts thanks to auxotrophy markers URA 3 and ADE 2 which complement the corresponding deficient genes in the yeast genome. The addition of the auxotrophy marker ADE 2 allows a better stability of the vector when the transformed yeasts are cultured in a culture medium that becomes spontaneously limiting to high cell density adenine. The plasmid can multiply in bacteria thanks to an origin of replication of E. coli and in yeasts by means of a fragment of the origin of replication of plasmid 2.

 Transformation of the two W (hR) W (R) strains with the plasmid p1A1 The yeast transformation protocol, adapted from Gietz et al. (1992), initially requires the yeast to be competent. For this purpose, the W (R) and W (hR) strains cultured in YPGA medium (glucose 20 g / l, yeast extract 10 g / l, bactopeptone 10 g / l and adenine 20 mg / l) until to achieve an OD600nm between 0.5 and 2. After centrifugation (6000 g, 3 min, 20 C), the cell pellet is washed twice with 25 ml of sterile distilled water, then once with 2 ml of Tris buffer. 100 mM HCl, 1 mM EDTA, 0.1 M lithium acetate. The cell pellet is taken up in 1 ml of this same buffer. Then, in order to proceed with the transformation of the yeasts, are mixed the aliquot of the transforming vector p1A1, 50 g of denatured DNA of salmon sperm, 100 ml of competent yeasts and 300 l of a solution of PEG-4000 to 40% in 100 mM Tris-HCl buffer, 1 mM EDTA, 0.1 M lithium acetate The mixture is incubated for 30 min at 28 ° C. and then 15 min at 42 ° C. After centrifugation (11000 g, 3 min,

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 C), the cell pellet is taken up in 100 l of Ringer's liquid. The 100 l are spread on SGI medium (glucose 20 g / l, yeast nitrogen base 7 g / l, bactocasamino acids 1 g / l, tryptophan 20 mg / l and agar 20 g / l). The dishes are incubated for 2 to 3 days at 30 ° C.

Similarly, the strain W (R) pP73Al results from the transformation of the strain W (R) with the plasmid pV60 in which the gene of the cinnamate 4-hydroxylase of Jerusalem artichoke or P450 73A1 (plasmid called p73Al - see Figure 3) has been inserted (Urban et al., 1994b).

EXAMPLE 3 Example of a digestion protocol in the digestive systems TIM1 and TIM2 (modified protocol of WO 94/09895) A Digestion in the artificial stomach and small intestine or TIM 1 (see FIG. 1) The digestion time is programmed as the case may be, at 240 or 360 minutes.

Solutions used during digestion Various solutions simulating stomach and intestinal secretions are prepared and stored in ice for those containing enzymes. All solutions are prepared with demineralised water.

the solutions simulating the secretions of the stomach: the gastric juice: NaCl 53 mM, KCl 15 mM, CaCl 2 1 mM and NaHCO 3 7mM, pH 4, to which we add pepsin at a rate of 588 U / ml, * 1M HCl or water so that the pH follows in the stomach a predetermined curve.

- solutions simulating the secretions of the small intestine: * 7% pancreatin solution, * 4% bile solution (for the first 30 minutes of digestion) and 2% (for the rest of digestion) 1X electrolyte solution: 86 mM NaCl, 8 mM KCl and 2 mM CaCl 2, 1 M NaHCO 3 or IX electrolyte solution to maintain the pH at 6.5 at the duodenum, 6.8 at the jejunum and 7.2 at the ileum.

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Digestion Prior to the food, the residues simulating what remains on average in a fasting individual are introduced into the various compartments of the digestive system: the gastric residue (gastric juice adjusted to pH 2 and preincubated for 15 min at 37 ° C.), the duodenal residue (bile 2%, pancreatin 1.75%, 0.25X electrolyte solution of the small intestine and trypsin 7773 U / ml, preincubated 10 min at 37 C) and the jejunal and ileal residues (electrolyte solution IX of the small intestine).

The dialysis fluids for the jejunum (1.55% bile, 8 mM KCl, 86 mM NaCl) and the ileum (IX electrolytes) are maintained at 37 ° C. during the digestion time and circulate at a flow rate of 10 ml / min (dialysis fibers: HG-400, threshold = 8000 Da).

The yeast strain studied, resuspended in 300 ml of semi-skimmed milk or YPL culture medium (yeast extract 10 g / l, bactopeptone 10 g / l, galactose 20 g / l), is introduced into the stomach. The yeasts are prepared as described in Example 4.

Kinetics The solutions simulating the different secretions are delivered according to a predetermined kinetics according to the parameters in vivo: * in the stomach: the gastric juice is delivered at a flow rate of 0.25 ml / min, the HCl and the water are introduced at a total flow rate of 0.25 ml / min.

in the duodenum: the 7% pancreatin solutions are delivered at a flow rate of 0.25 ml / min, the bile solution (at 4 or 2%) at a flow rate of 0.5 ml / min, the 1 M NaHCO 3 and the IX electrolyte solution at a total flow rate of 0.25 ml / min.

in the jejunum and the ileum: the 1M NaHCO 3 is introduced at a flow rate of 0.25 ml / min, if necessary.

The gastric and ileal emptying curves, modeled by the formula f = 1-2- (t / t1 / 2) ss have for parameters: t1 / 2 = 30 min; B = 1 and t1 / 2 = 160 min; ss = 1.6.

B Fermentation in the artificial colon or TIM 2 (see Figure 2) After a phase of stabilization of the flora of 2 days, the fermentation is maintained 60 hours for the experiment.

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Seeding of the colon All the steps of the seeding are carried out under nitrogen flow.

200 g of fresh faeces (from healthy volunteers) are mixed in 200 ml of 0.1 M phosphate buffer at pH 6.5, autoclaved beforehand and placed under a stream of nitrogen for at least 2 hours. The mixture obtained is filtered on sterile gauze. The filtrate is centrifuged (25000 g, 30 min, 4 C). The bacterial pellet and the mucus are taken up in the base medium (Ileum delivery medium) itself diluted 10 times in 0.1M phosphate buffer.

About 10 g of filtration retentate are added to this bacterial solution in order to enrich it with fibers. The mixture is mixed again and then introduced into the colon using a syringe.

Solutions used during the fermentation Various solutions involved in the constitution of the nutrient medium of the faecal flora and the dialysis solution of the colon are prepared, autoclaved for 15 min at 120 ° C. and then stored at 4 ° C. until use (except the vitamin solution). which is sterilized by filtration and stored at -20 ° C). All solutions are prepared with demineralised water.

a solution of electrolytes 10 X: heme 0.1 g / l, bile salts 0.5 g / l, cysteine 4 g / l, FeSO 4 0.05 g / l, CaCl 24.5 g / l, MgSO 4 5 g / l 1, NaCl 4.5 g / l and K2HPO4 25 g / l, - Carbohydrate solution X: pectin 12 g / l, xylan 12 g / l, arabinogalactan 12 g / l, amylopectin 12 g / l and starch 100 g / l, - a solution containing lipids and proteins (TBC 40X): 80 80 ml / l, bactopeptone 120 g / l and casein 120 g / l, - a solution of vitamins 10 X: 10 mg / 1, biotin 20 mg / l, vitamin B 12 5 mg / l, pantothenate 100 mg / l, nicotinamide 50 mg / l, p-aminobenzoic acid 50 mg / l and thiamine 40 mg / l.

Stabilization of the flora and fermentation The dialysis solution (1 X electrolyte solution of the colon and 0.01 X vitamin solution, pH 6.5) is bubbled under nitrogen flow overnight. It is delivered at a rate of 1 ml / min for the duration of the stabilization and

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 fermentation (dialysis fibers: JFM M5ID X Flow).

"Ileum delivery medium" (1 X colonic electrolyte solutions, 10 X carbohydrate solution, 10 X TBC, 0.01 X vitamin solution and 20 g / l mucus, pH 6.5), simulating the product of digestion from the ileum, is introduced into the colon at a rate of 3 ml per hour.

The yeast strain studied, resuspended in 5 ml of distilled water, is introduced into the colon after stabilization of the flora. The yeasts are prepared as described in Example 4.

EXAMPLE 4 Monitoring of recombinant W (R) pHlAl and W (hR) pHlAl yeasts in artificial digestive systems Preparation of yeasts The W (R) pHlAl and W (hR) pHlA1 strains are cultured in SGI minimum medium (glucose 20 g / l, yeast nitrogen base 7 g / l, bactocasamino acids 1 g / l and tryptophan 20 mg / l) up to a concentration of 106-107 cells / ml.

The culture is then centrifuged in sterile plastic tubes (5000 rpm, 10 min, 4 C). The supernatant is discarded and the cells are washed in 15 ml of physiological saline (NaCl 9 g / l).

After centrifugation (5000 rpm, 10 min, 4 C), the supernatant is discarded and the cell pellet is resuspended in the feed.

The yeasts are introduced into the TIM 1 resuspended in 300 ml of semi-skimmed milk or are introduced into the TIM2 resuspended in 5 ml of distilled water.

Enumeration of yeasts - Collection of samples in TIM 1: ml of the starting food (containing the yeast strains) is taken before introduction into the stomach (t = 0), then 1 ml is taken at times 120,240 and 360 min. in ileal effluents collected over time intervals 0-120 min, 120-240 min, 240-360 min, - Specimen collection in TIM 2: 1 ml of colonic content is collected before

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 seeding of the colon (t = ofec) as well as 1 ml of the suspension of yeasts before introduction into the colon (t = 0), then 1 ml of the colonic contents is taken every 12 hours in the colon (t = 1, 12, 24, 36.48 and 60 h), - Yeast culture: the samples are diluted in physiological saline and spread on SGI medium. Ampicillin (100 g / ml) is added to the medium to remove contaminants from the digestive system and / or faecal flora, as well as ethanol (3% v / v) to promote yeast development.

- Enumeration: the enumeration of the dishes is carried out after 2 to 3 days of incubation at 30 C.

Results: The experiments carried out made it possible to demonstrate the viability in the artificial digestive systems of recombinant S cerevisiae W (hR) pH1A1 and W (R) pH1Al strains introduced simultaneously into the stomach or into the colon.

50% 18.9 (n = 3) of the recombinant yeasts introduced into the stomach are found at the outlet of the ileum after 6 h of digestion (Figure 4).

The yeast survival rate in the colon appears to be lower since, after 12 hours of fermentation, only 4% of the introduced yeasts (n = 1) are found (FIG. 5).

The causes of this sharp decrease in the survival rate in the colon remain indeterminate, especially since, after one hour of fermentation, all the yeasts introduced were still present in the colon. Two hypotheses have been put forward questioning the competition exerted by the faecal flora and the conditions of quasi anaerobiosis reigning in the artificial colon.

Yeast identification by Polymerase Chain Reaction (PCR) The PCR technique had to be optimized for the monitoring of recombinant yeasts in the digestive environment.

The PCRs can be carried out from colonies isolated on a Petri dish or yeasts put back into liquid culture. In the first case, a pinhead is taken from a colony which is resuspended in 10 ml of distilled water before boiling for 10 minutes, in the second case the culture must be in phase. exponential

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 growth (OD 600 nm between 0.5 and 2). After centrifugation (10000 g, 10 min, 4 C), the cell pellet is taken up in 50 l of distilled water and boiled for 10 min.

In liquid culture, the best results (in terms of amplification intensity and reproducibility of the experiments) are obtained when the yeasts are in the exponential phase of growth (OD600nm of 0.5 to 2), whereas on solid medium the stage of Yeast growth does not seem to influence the results.

The primers used are either internal to the genes (human and yeast P450 1A1 and NADPH-cytochrome P450 reductases), or located both on the gene and its GAL10-CYC1 promoter in order to verify the stability of this gene (FIG. 3). . They are chosen so that they hybridize as little as possible about themselves, with themselves and with each other by using the program on the Internet at the following address: http: // www. Williamstone.com/primers/calculator/calculator. The PCR samples contain 1X PCR buffer, 1.5 mM MgCl 2, 4 dXTP 0.2 mM, 5 'and 3' 0.5 Il M primers, 25 U / ml Taq polymerase and 0.06% DNA.

The PCR program used is as follows (30 cycles): 5 min 95 C, denaturation 30 s 95 C, initiation 30 s 51 C, synthesis of DNA 2 min 72 C and final extension 10 min 72 C.

After PCR, the samples are mixed with a stop solution (bromophenol blue 0.25%, Ficoll 10% and SDS 1%) and deposited on a 1.5% agarose gel containing BET 0.5 μg / ml. . The migration is carried out in an electrophoresis tank containing 40 mM Tris-base, 20 mM glacial acetic acid, 1 mM EDTA buffer, for 15 minutes at 100V. After migration, the DNA is visualized under UV.

Results: The yeast strains were identified by PCR both at the exit of the small intestine until 6 h after their introduction in the stomach and at the exit of colon until 48 H after their introduction in this one (Figure 6).

EXAMPLE 5 Measurement of the Activity of the Recombinant Yeasts After Passage Into TIM1 (W (hR) pH1Al) and In Situ (W (R) pP73Al)

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 1 Measurement of recombinant yeast activity W (hR) pHlAl after pass! in TIM1 The strain W (hR) pH1Al is used to demonstrate the maintenance of the activity of recombinant yeasts after passage through TIM1.

P450 1A1 has the ethoxyresorufin-O-deethylase (EROD) activity and catalyzes the conversion of 7-ethoxyresorufin to resorufin.

Yeast culture The yeasts are precultured for 12 hours at 28 ° C., with stirring, in 30 ml of SGI (glucose 20 g / l, yeast nitrogen base 7 g / l, bactocasamino acids 1 g / l and tryptophan 20 mg / l). Then, a culture of 48 hours at 28 ° C., with stirring, is carried out in 250 ml of YPGE (glucose 20 g / l, yeast extract 10 g / l, bactopeptone 10 g / l and ethanol 3%).

During the last 12h, induction of P450 1A1 is caused by adding galactose (25 ml of a 200 g / l solution). The yeast culture is then either used as such for the determination of EROD on cells, or undergoes various treatments to prepare microsomes (Pompon et al., 1996).

Content of P450 1A1 The P450 1A1 content was measured on microsomes of strain W (hR) pH1A1 after passage through the digestive systems.

For this, we use the method of Omura and Sato (1964), based on the property of the hemoprotein to have a maximum absorption around 450 nm when it is reduced and linked to carbon monoxide. The microsomal suspension is taken up in 50 mM Tris-HCl buffer, 1 mM EDTA, pH 7.4 in order to obtain an OD 600 nm of approximately 0.8, and then reduced by a spatula tip of sodium dithionite. The absorption spectrum is recorded before and after bubbling CO into the suspension. The concentration of P450 1A1 (only detectable P450) is calculated according to the Beer-Lambert law (E448 nm = 91 nM-1ch-1).

The microsomal proteins are assayed with the Protein Assay ESL kit (Boehringer Mannheim).

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Determination of EROD activity The enzymatic activity ethoxyresorufin O-deethylase (EROD) is characteristic of cytochrome P450 1A1. It is measured on strain W (hR) pHlAl, after passage through artificial digestive systems. This activity is assayed according to the fluorimetric method described by Burke et al. (1985) which measures the increase in fluorescence related to the formation of resorufin, the dealkylation product of 7-ethoxyresorufin.

The EROD activity is measured on microsomes or on cells at 24 ° C. In the first case, 1 ml of 50 mM Tris-HCl buffer, 1 mM EDTA, pH 7.4.5 l of NADPH (10 mg / ml) and 2 l of a solution of 7-ethoxyresorufin saturated in ethanol are mixed. After adding the microsomes, the increase in fluorescence (#ex = 530 nm, #em = 586 nm) is recorded. In the second case, after centrifugation (11000 g, 30 s, 4 C) of 1 ml of culture at an OD600nm of 3 minimum, the cell pellet is taken up in 1 ml of 50 mM Tris-HCl buffer, 1 mM EDTA, pH 7.4. The increase in fluorescence (, ex = 530 nm, #em = 586 nm) is recorded following the addition of 2 l of a solution of ethanol-saturated 7-ethoxyresorufin.

Results: The W (hR) pH1Al strain still has the EROD activity, characteristic of P450 1A1, at the end of the small intestine and in the colon, that it is assayed on microsomes (approximately 20 pmol of resorufin per ml of microsomal suspension per minute) or on cells (approximately 5 pmol of resorufin per ml of culture per minute).

2 Measurement of recombinant W (R) pP73Al yeast activity in situ (in TIM1) The yeast strain used to demonstrate the activity in situ is the strain W (R) pP73Al.

P450 73A1 has cinnamate 4-hydroxylase activity and catalyzes the conversion of trans-cinnamic acid to p-coumaric acid.

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Preparation of yeast The yeasts are precultured for 12 hours at 28 ° C., with stirring, in 30 ml of SGI (glucose 20 g / l, yeast nitrogen base 7 g / l, bactocasamino acids 1 g / l and tryptophan 20 mg / l). Then, a culture of 48 h at 28 ° C., with stirring, is carried out in 250 ml of YPGE (glucose 20 g / l, yeast extract 10 g / l, bactopeptone 10 g / l and ethanol 3%) until it reaches a density of 7-8x10 lev / ml.

During the last 12 hours, the induction of CYP73A1 (gene coding for P45073A1) is caused by the addition of galactose (25 ml of a 200 g / l solution) until a density of 2 × 10 8 lev / ml is reached.

The culture is then centrifuged in sterile plastic tubes (5000 rpm, 10 min, 4 C). The supernatant is discarded and the cells are washed in 15 ml of physiological saline (NaCl 9 g / l).

After centrifugation (5000 rpm, 10 min, 4 C), the supernatant is discarded and the cell pellet is resuspended in the feed (300 ml of YPL medium).

Sequence of digestion The substrate (trans-cinnamic acid) is introduced extemporaneously in the stomach at the concentration of 670 M. The digestion is carried out over 240 minutes.

Samples (1 ml) are taken at different levels of the digestive tract: in the food before introduction into the system, in the stomach after 15.30 and 45 minutes of digestion, in the duodenum after 15.30. , 45.60, 75 and 90 min of digestion, in the jejunum after 45.60, 90.120, 150 and 180 min of digestion, in the ileum after 60.90, 120.180 and 240 min of digestion, in cumulative outputs over 0-60 min, 60-120 min, 120-180 and 180-240 min at 60,120, 180 and 240 min and in cumulative uptake on 0-60,60-120, 120-180 and 180- 240 to 60.120, 180 and 240 min.

Sample processing 100 μl of trifluoroacetic acid (25% in water w / v) is added to 1 ml of sample to stop the reaction. After microcentrifugation (3 min, 10000 rpm, temperature

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 ambient), the supernatant is recovered and filtered (0.45 μm). Samples are stored at -20 C before analysis.

HPLC analysis After filtration, 10 l of the samples are injected into the Merck Lichrospher 100 RP-18 column (5 m, 12.5 cm * 4 mm). A gradient between 2 phases A and B is carried out over 34 min with a flow rate of 1 ml per minute, as described below: Phase A: Water 95, methanol 5 and acetic acid 0 1 (v / v), Phase B: Acetonitrile 95, Methanol 5 and acetic acid 0 1 (v / v), 0-16 min: 90% A, 10% B, 16-18 min: linear gradient up to 80% A, 20% B, 18-32 min: 80% A, 20% B, 32-34 min: linear gradient up to 90% A, 10% B.

The detection is carried out by spectrophotometry at 314 nm between 0 and 16 min, then at 280 nm between 16 and 34 min. The retention time of the compounds is 60 min. For p-coumaric acid and 28.degree. min for trans-cinnamic acid Results The enzymatic activity cinnamate 4-hydroxylase was detected in all the digestive compartments of TIM 1 (stomach, duodenum, jejunum and ileum) The results are presented in FIG. 7. The molar ratio of p-coumaric acid / mol of trans-cinnamic acid increases along the digestive tract, proving that recombinant yeasts are active in all compartments of TIM 1 If a balance is made after 4 hours of digestion - have were introduced in TIM 1. 9.6 * 109 cells of yeast and 205.4 mol of trans-cinnamic acid, - were found at the end of digestion (ie 240 minutes after the beginning of the digestion): 8.9 * 109 cells of yeasts (in output of the ileum), 53.6 mol of acid

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 p-coumaric (in the entire digestive system) and 56.1 pmol of trans-cinnamic acid

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Claims (5)

1. microorganisms comprising at least one heterologous DNA fragment comprising a gene of interest capable of expressing itself in the digestive environment, characterized in that its survival rate is greater than 25%, 50%, 75 %, preferably 80% at the outlet of the ileum after 6 hours of digestion and is greater than 2%, 4%, preferably 8% after 12 hours of fermentation in the colon 2 Microorganisms according to claim 1 characterized in that that the heterologous DNA fragment consists of an expression cassette comprising at least one gene of interest, a promoter and a terminator. Microorganisms according to one of the preceding claims, characterized in that they are yeasts selected in particular from Saccharomyces cerevisiae, S boulardii, S uvarum, Hansenula anomala, Schizosaccharomyces. pomhe and Klevuromyces lactis. Microorganisms according to one of the preceding claims, characterized in that the gene of interest encodes a product selected from peptides of interest for vaccination, peptides of hormonal interest, peptides of therapeutic interest, peptides with an antibiotic effect, peptides capable of trapping or complexing molecules, enzymes or any peptide having bioconversion or biosynthesis activities, in particular detoxification enzymes Microorganisms according to one of the preceding claims, characterized in that the gene of interest code for an enzyme of xenobiotic metabolism selected from the phase 1 enzymes (P450 1A1, IA2, IB1, 2B6.2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5 and 73A1), the environment enzymes of phase 1 (cytochrome P450 reductase and cytochrome b5) and phase II enzymes such as glutathione S-transferase (GST), N-acetyltransferase (NAT), <Desc / Clms Page number 43>  quinone reductase (QR), aldehyde dehydrogenase (ALDH) and uridine diphosphoglucuronosyl transferase (UGT) 6 Microorganisms according to one of the preceding claims, characterized in that the gene of interest is placed under the control of a promoter selected from constitutive or inducible promoters, strong promoters, promoters specifically active in a segment of the digestive tract under aerobic, semi anaerobic or anaerobic conditions, and promoters regulable by a molecule introduced into the intestinal medium. 7. A method for studying, controlling and / or selecting microorganisms according to one of claims 1 to 6 characterized in that it implements an artificial digestive system. 8. A method of study and / or control according to claim 7 comprising the steps of: a) adding a microorganism according to one of claims 1 to 6 in a liquid nutrient medium, b) introducing said medium to the entry of an artificial digestive system comprising at least one of the compartments, c) incubating and counting and / or testing the activity of the gene of interest after passage through the digestive or in situ systems. selection system according to claim 7 comprising the steps of a) adding microorganisms according to one of claims 1 to 6 in a liquid nutrient medium, b) introducing said medium to the inlet of an artificial digestive system comprising at least minus one of the compartments, c) incubate and recover the surviving microorganisms after passage through said system; d) Repeat steps a) to c) until a population is obtained <Desc / Clms Page number 44>  homogeneous micro-organisms adapted to the digestive environment and capable of effectively expressing the gene of interest. Microorganism obtained from the method according to one of claims 7 to 9 11. Microorganism according to claim 10 characterized in that its survival rate is greater than 25%, 50%, 75%, preferably 80%. % at the outlet of the ileum after 6 hours of digestion and is greater than 2%, 4%, preferably 8% after 12 hours of fermentation in the colon. 12. Microorganism according to one of claims 10 and 11 characterized in that it is a yeast 13 Combination product comprising a microorganism according to one of claims 1 to 6 and 10 to 12 and an active substance or a precursor of an active substance for simultaneous, separate or spread use over time. 14 Product according to claim 13 characterized in that the microorganism acts on the active substance or vice versa. A pharmaceutical composition comprising microorganisms according to one of claims 1 to 6 and 10 to 12 being in a dosage form adapted for oral or rectal administration so as to route them intact to the target site of the digestive tract and to maintain their integrity at this level for a given time. Pharmaceutical composition according to claim 15, in which the microorganisms, in particular the yeasts, are protected by a coating and / or a membrane of the resin and / or polymer type forming spheres, microspheres, granules or microgranules. <Desc / Clms Page number 45>  17. Pharmaceutical composition according to claim 16 characterized in that it is in the form of capsules, tablets, or dragees. 18. Use of microorganisms according to one of claims 1 to 6 and 10 to 12 for the preparation of a drug 19. Use according to claim 18 for the preparation of detoxifying agents, metabolic correction agents or agents capable of modifying the intestinal flora. 20. Use according to claim 18 for the preparation of a medicament for reducing the toxicity and / or increasing the bioavailability of an active substance or its precursor 21. Use according to claim 18 for the preparation of a medicament capable of provide in situ active peptides, peptides of vaccine interest, peptides of hormonal interest, peptides of therapeutic interest, peptides with antibiotic effect or peptides capable of trapping or complexing molecules