Pro-resolving pharmaceutical preparation in gel form

Abstract

The invention relates to a pharmaceutical preparation comprising a supernatant obtained from a co-culture of irradiated leukocytes and phagocytes, characterized in that it further comprises a polymer selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hyaluronic acid (HA), carboxymethylcellulose (CMC) and polycarbophil (PC) or a mixture thereof (HPMC), in which the weight content of the polymer is selected so as to confer a gelled form on said pharmaceutical preparation.

Description

[0001] Description

[0002] Title of the invention: PRO-RESOLVING PHARMACEUTICAL PREPARATION IN GEL FORM

[0003] The invention relates to a pharmaceutical preparation comprising a supernatant obtained from a coculture between phagocytes and irradiated leukocytes.

[0004] An inflammatory reaction (or inflammation) is a biological process involved in the body's natural defense reactions but also associated with a multitude of pathological reactions.

[0005] Thus, an inflammatory disease is generally due to prolonged and unresolved inflammation within a specific organ or part of the human or animal body. Such a disease can be caused by various genetic, immunological, infectious, or cancer-related factors. Therapeutic treatment of an inflammatory disease is traditionally based on targeting symptoms using so-called anti-inflammatory drugs.

[0006] There are known inflammatory and degenerative diseases in which unresolved inflammation appears to be the cause of chronic inflammatory diseases. Indeed, inflammation is a natural reaction that self-regulates and stops through the biological process called inflammation resolution.

[0007] The resolution of inflammation involves various cellular partners and molecular factors and results in the cessation of the inflammatory response and the repair / regeneration of damaged tissues. A disruption of the resolution process, on the other hand, leads to uncontrolled inflammation, persisting at high or low levels, locally or systemically, and promoting the development of chronic or degenerative inflammatory pathology.

[0008] One of the key mechanisms of the resolution process is the release of pro-resolving factors by phagocytic cells eliminating dying cells from the sites of the inflammatory reaction. Thus, these factors have the capacity to stop the ongoing inflammatory reaction and initiate tissue repair and regeneration. Among the set of pro-resolving factors, the prior art describes in particular in the Applicant's document EP 3 810 157, a subset of factors called "resolutome" capable of triggering the resolution of inflammation.

[0009] EP 2 941 257 describes a pharmaceutical preparation for use in the prevention or treatment of a pathological immune response. This pharmaceutical preparation comprises a supernatant, sometimes called a "secretome", obtainable from the coculture of phagocytes with apoptotic cells. The application of this pharmaceutical preparation is for the treatment of a pathology such as an autoimmune disease, for example rheumatoid arthritis (RA), chronic inflammatory bowel diseases (IBD) or tissue regeneration.

[0010] The state of the art has thus demonstrated that pro-resolving factors, or a resolutome, produced by phagocytes eliminating dying cells, have a therapeutic activity that reactivates the resolution process that is defective in the context of inflammatory pathologies. Under certain conditions, this can allow the disease to be stopped.

[0011] However, the full range of therapeutic effects in different pathological situations could only be observed through intravenous administration of pro-resolving factors. This poses problems, particularly for the specific targeting of local tissue inflammation.

[0012] Indeed, localized tissue inflammation is found, for example, in the pathologies of Crohn's disease or inflammatory ulcerative colitis. In these diseases, inflammation particularly affects the intestinal mucosa. The state of the art, which uses a systemic approach, is not optimal for the treatment of this type of localized tissue inflammation.

[0013] Furthermore, the production of pro-resolving factors of the secretome and / or resolutome type is complex and expensive. There is a need to reduce the dosage and / or the therapeutically effective quantity of these factors.

[0014] The present invention improves the situation.

[0015] Thus, the invention relates to a pharmaceutical preparation comprising a supernatant obtained from a coculture between phagocytes and irradiated leukocytes. The pharmaceutical preparation of the invention further comprises a polymer selected from the group consisting of hydroxylpropylmethylcellulose (HPMC), hyaluronic acid (HA), carboxymethylcellulose (CMC) and polycarbophil (PC) or a mixture thereof.

[0016] The invention makes it possible in particular to reduce the quantity of supernatant necessary to obtain a therapeutic effect. In other words, the combination of the supernatant active ingredient with one or more of the polymers chosen from hydroxypropylmethylcellulose (HPMC), hyaluronic acid (HA), carboxymethylcellulose (CMC) or polycarbophil (PC), makes it possible to lower the quantity of supernatant necessary to be therapeutically effective. A synergistic effect is observed by combining a supernatant and a polymer of the invention.

[0017] In addition, the polymer used in a predefined quantity by weight (concentration or %) depending on the nature of the polymer chosen, results in the pharmaceutical preparation of the invention in gel form. An advantage of the preparation in gel form is that it can be administered locally in a targeted manner.

[0018] More generally, the invention relates to the addition of polymers making it possible to increase the viscosity and adhesion of therapeutic products of the secretome or resolutome type, in order to be able to apply it directly to an affected mucosa. The invention thus makes it possible to target the affected inflammatory area, increase the residence of the product on this area and obtain a therapeutic effect at least identical but with a lower quantity of product compared to intravenous systemic injection. In particular, the invention makes it possible to target the inflammatory area and to exert the stopping of tissue inflammation and to trigger the repair of the damaged area. The invention allows in particular the treatment of the intestinal mucosa during inflammatory colitis.

[0019] In one embodiment, the weight content of the polymer in the pharmaceutical preparation is chosen so as to impart a gelled form to said pharmaceutical preparation.

[0020] In another embodiment, the polymer is hydroxypropylmethylcellulose (HPMC). Preferably, the HPMC content is between 0.5% and 1.5% by weight. In a first particularly preferred embodiment, the HPMC content is 0.5% by weight. In a second particularly preferred embodiment, the HPMC content is 1.5% by weight.

[0021] In the embodiment in which the polymer is hyaluronic acid (HA) its content is preferably 0.5% by weight. In the embodiment in which the polymer is carboxymethylcellulose (CMC) its content is preferably 0.4% by weight. In the embodiment in which the polymer is polycarbophil (PC) its content is preferably 0.1% by weight.

[0022] In one embodiment, the pharmaceutical preparation of the invention is for use as a medicament.

[0023] In a particular embodiment, the pharmaceutical preparation of the invention is for use in the treatment of inflammatory and degenerative diseases.

[0024] In another particular embodiment, the preparation of the invention is for use in the treatment of inflamed colonic mucosa during chronic inflammatory bowel disease. The chronic inflammatory bowel disease may be inflammatory colitis, Crohn's disease (CD) or ulcerative colitis (UC). More generally, the pharmaceutical preparation of the invention is for use in the treatment of mucosal inflammation.

[0025] In a preferred embodiment, the supernatant of the pharmaceutical preparation is Resolvix®. The product Resolvix® is available from the Applicant.

[0026] Other advantages and characteristics of the invention will appear on reading the detailed description below and on the attached drawings in which:

[0027] Figure 1 shows a graphical representation of the evolution of the viscosity of a supernatant of the invention (i.e. obtained from a coculture between phagocytes and irradiated leukocytes) as a function of the addition of different polymers;

[0028] Figure 2 shows graphical representations of the pro-resolving effect of a supernatant of the invention in the presence of polymers on wound healing on MODE-K and Caco-2 cells; and Figure 3 shows a diagram of the anti-inflammatory effect of a supernatant of the invention in an in vitro intestinal inflammatory model;

[0029] Figure 4 shows a graphical representation of the therapeutic effect of a supernatant of the invention in an experimental model of inflammatory colitis;

[0030] Figure 5 shows diagrams of the reduction of inflammatory biomarkers associated with acute inflammatory colitis following different treatments using the pharmaceutical preparation according to the invention;

[0031] Figure 6 shows graphical representations of the therapeutic effect by the clinical scores of chronic inflammatory colitis, as well as the survival after treatments with the pharmaceutical preparation according to the invention in gel form; and

[0032] Figure 7 shows diagrams of the reduction of inflammatory biomarkers associated with chronic colitis following treatments with the pharmaceutical preparation according to the invention.

[0033] The figures, tables and description below contain, for the most part, elements of a certain nature. The figures and tables are an integral part of the description, and may therefore not only serve to better understand the present invention, but also contribute to its definition, where appropriate.

[0034] The pharmaceutical preparation of the invention comprises a supernatant obtained from a coculture between phagocytes and irradiated leukocytes. A supernatant suitable for carrying out the present invention is available from the Applicant under the reference Résolvix®. Document EP3810157B1, to which the reader is invited to refer, describes in detail the product Résolvix® as well as its process for obtaining it.

[0035] According to one embodiment, a method for obtaining a supernatant may comprise the following steps: (i) generating monocyte-derived phagocytes from blood leukocytes, (ii) bringing these phagocytes into contact with previously irradiated blood leukocytes, in particular by X-rays, (iii) coculturing the phagocytes with the irradiated leukocytes for a predetermined time, and (iv) collecting factors contained in the supernatant produced by the cells in coculture. A preferred embodiment for obtaining the supernatant of the invention is described below.

[0036] Leukocytes are isolated (JO) by density gradient from buffy coats of whole blood. The leukocytes are washed and then resuspended in a defined culture medium, preferably known as RPMI 1640. Other culture media can be used, particularly those based on salts, amino acids and vitamins. The culture medium containing the leukocytes is then divided into two bags: 60% in a first bag and 40% in a second bag.

[0037] The first pouch receives a differentiation factor, preferably Macrophage Colony-Stimulating Factor (M-CSF). The first pouch is stored under conditions that preserve cell viability, for example at 37°C, 5% CO2, for several days, preferably 3 days, and then receives fresh culture medium again. The first pouch is left as is, which allows the production of phagocytes after several days of storage, preferably 4 days, under conditions that preserve cell viability (for example at 37°C, and 5% CO2).

[0038] The second bag is stored at low temperature, preferably below +10°C, and even more preferably below +5°C. On the seventh day (D7), the second bag is irradiated, preferably by X-rays.

[0039] The respective culture media of the first and second bags are then removed. Then, a secretion medium is added to the second bag. The secretion medium contains in particular amino acids and vitamins. Preferably, the secretion medium is of the Minimum Essential Medium (MEM) type. The entire contents of the second bag are then transferred to the first bag for storage under conditions preserving cell viability (37°C, 5% CO2 for example).

[0040] During this storage, coculture takes place, resulting in the production of a conditioned supernatant, which can take several days, preferably 2 days. In the present description, the term "conditioned supernatant" means a supernatant containing cell-derived factors. At the end of this coculture period, the supernatant is collected in a new bag, filtered and then stored at a very low temperature, preferably around -80°C. The supernatant is then advantageously mixed with other supernatants produced, according to the method described above, from buffy coats, then this mixture is divided into unit doses, lyophilized or not, and then stored.The person skilled in the art knows how to apply to the process described here all the precautions to meet the so-called "good manufacturing practice" criteria governing the production and release of complex human biological medicinal products derived from blood defined by the European Medicines Agency.

[0041] In one embodiment, the supernatant of the invention comes from a mixture of supernatants obtained according to the production method described above from several leukocyte sources.

[0042] The present invention combines a Résolvix® type supernatant with a polymer. The polymer may be chosen from polysaccharides, polyacrylates or cellulose polymers. Among the polysaccharides, mention may be made of linear heteropolysaccharides, including cellulose derivatives, such as hydroxypropylcellulose (or HPMC, which is non-ionic), carboxymethylcellulose (or CMC which is anionic), and other polysaccharides, such as gellan gum (anionic) and hyaluronic acid (anionic). Among the polysaccharides, mention may also be made of branched heteropolysaccharides, including in particular xanthan gum (anionic). Among the polyacrylates (also called acrylic acid polymers), mention may be made of polycarbophil (anionic).

[0043] Combining the Résolvix® type supernatant with one or more polymers increases the viscosity of the supernatant (sometimes called "Resolutome"). Preferably, the viscosity increase is carried out so as to obtain a gelled supernatant, i.e. in the form of a gel.

[0044] Figure 1 shows a graphical representation of the viscosity of Resolvix® in the presence of different polymers according to the shear rate. The viscosity (mPa per second) was evaluated according to the shear rate (s-1 ) using a Kinexus Pro+ rheometer (NETZSCH) in the different preparations of Resolvix® alone or combined (%m / V) at:

[0045] - 0.5% hydroxypropyl methylcellulose (HPMC) (1X);

[0046] - 1.5% (3X) hydroxypropyl methylcellulose (HPMC);

[0047] - 0.5% hyaluronic acid (HA);

[0048] - 0.4% carboxymethylcellulose (CMC);

[0049] - 0.3% gellan gum (GG);

[0050] - 0.4% xanthan gum (GX); and

[0051] - 0.1% polycarbophil (PC).

[0052] The data in Figure 1 are from 3 independent experiments represented as mean + / - standard error of the mean and show that the addition of a polymer to Résolvix®, apart from the addition of PC, allows to obtain a viscosity statistically higher than the Résolvix® alone condition according to the shear rate - according to the one-way analysis of variance (ANOVA) followed by a Tukey multiple comparison test. The combination of Résolvix® with 0.1% PC does not allow to obtain gelation of Résolvix®. At higher PC concentrations, the product did not show superior gelation (and on the contrary a lower healing power, see below). More specifically, it seems that the increase in PC concentration does not allow to increase the viscosity because there is an ionic interaction with the salts of the medium which disfavors gelation.Furthermore, at a PC level of approximately 0.5% a negative effect on healing was observed.

[0053] In a preferred embodiment, the pharmaceutical preparation of the invention is in gel form.

[0054] The Applicant studied the pro-resolving effect of the product Résolvix® in combination with different polymers.

[0055] Figure 2 shows graphic representations of the pro-resolving, here pro-healing, effect of Résolvix® mixed or not with polymers on the filling capacity of a wound made on a monolayer of MODE-K (Figure 2a) or Caco-2 (Figure 2b) cells. Resolvix® alone or gelled with different polymers, i.e. hydroxypropyl methylcellulose (HPMC) at 0.5% (1 X) or 1.5% (3X), hyaluronic acid (HA) at 0.5%, carboxymethylcellulose (CMC) at 0.4%, gellan gum (GG) at 0.3% or xanthan gum (GX) at 0.4% or polycarbophil (PC) at 0.1%, was added onto a monolayer of MODE-K (Figure 2a) and Caco-2 (Figure 2b) cells at confluence (i.e. covering the entire culture surface), after making a wound along the entire culture surface, thus creating a wound to be filled.The wound-filling potential of cells in the presence of different combinations of Resolvix® alone or mixed with polymers was evaluated regularly for 40 hours. The data in Figure 2 (Figures 2a and 2b) are cumulative and derived from 3 independently performed experiments and represented as an average. All conditions are statistically different from the MEM control condition. The Resolvix®+CMC, +GX and +GG conditions are also statistically different from the Resolvix® alone condition (according to one-way analysis of variance (ANOVA) followed by a Tukey multiple comparison test).

[0056] Thus, the Applicant discovered, not without surprise, that the addition of the various polysaccharides, polyacrylates or cellulose polymers does not alter the pro-resolving effect of Résolvix®. In particular, Résolvix® combined with certain polymers retains its pro-healing properties both on human epithelial cells (Caco-2) and on rodent epithelial cells (MODE-K). The pro-healing properties are particularly retained with hydroxypropyl methylcellulose (HPMC) and hyaluronic acid (HA).

[0057] Combinations of Resolvix® with HPMC and HA were retained to evaluate pro-resolving properties in controlling inflammation using a tripartite in vitro model mimicking intestinal mucosal inflammation.

[0058] Figure 3 shows a histogram of the anti-inflammatory effect of Resolvix®, alone on the one hand, and gelled with hydroxypropyl methylcellulose or with hyaluronic acid on the other hand, in an in vitro intestinal inflammatory model.

[0059] Resolvix® alone and its gelled version with the different polymers, namely with 0.5% hydroxypropyl methylcellulose (HPMC-1X) and with 0.5% hyaluronic acid (HA), was added to culture wells containing leukocyte and epithelial cells separated by a hemipermeable membrane with 5 µm pores (CORNING™), for 24 hours at 37°C in a controlled atmosphere (5% CO2). At the end of culture, the cytokines TNF-a, IL-10, IL-6 and IL-8 were quantified by ELISA in the culture supernatant. Cumulative data from 3 independent experiments represented as mean + / - SEM. All "stim" conditions show for each cytokine a statistically increased quantity compared to the "no stim" control condition. The +Resolvix, +Resolvix / HPMC-1X and +Resolvix / HA conditions show significantly reduced quantities of cytokines compared to the "stim" condition.Significance was determined by statistical tests, two-way analysis of variance followed by Tukey's multiple comparison test.

[0060] The results show that Resolvix® gelled with HPMC or HA allows a drastic reduction of inflammation associated with leukocyte cells (TNF-a and IL-1 P) and epithelial cells (IL-6 and IL-8).

[0061] The invention therefore makes it possible to preserve the therapeutic effect of Résolvix®, in particular its pro-resolving properties, notably regenerative and restorative, in vitro.

[0062] Surprisingly, beyond the preservation of the therapeutic effect, a gain in efficacy is observed in an experimental murine model of inflammatory colitis. The model used allows the induction of inflammation of the intestinal mucosa by the addition of dextran-sodium-sulfate in the drinking water (3.5% weight / volume) of 8-week-old BALB / c mice, for 10 days, followed by a 3-day normal water cycle. Through the monitoring of the animals' daily clinical score, a clinical score taking into account weight loss, stool consistency and bleeding at the anus, the invention (Resolvix® combined with certain polymers) demonstrates a significant reduction in the clinical colitis score.Indeed, the product administered on the tenth day (D10), either in its standard version (intravenously, 1 mL of Résolvix® / animal), or in its gelled version (intrarectally, 100 pL / animal) with HPMC, HA, CMC or with an HPMC / PC mixture (PC alone does not allow satisfactory gelling of Résolvix® to be obtained, see Figure 1) allows a significant reduction in the clinical score compared to the control group, as shown in Figure 4 which shows a graphic representation of the therapeutic effect of Résolvix® in its known version and in its version according to the invention, i.e. in a gelled version using selected polymers, in an experimental model of inflammatory colitis.

[0063] The experiment lasted 13 days. Eight-week-old BALB / c mice received dextran sodium sulfate (DSS; 3.5% w / V) in their drinking water for 10 days and then unsupplemented drinking water for 3 days. On day 10, the mice received (black arrow in Figure 4) either 1 mL of Resolvix® (Colitis + Resolvix) or a control solution (PBS; Colitis + vehicle) intravenously, or 100 μL of Resolvix® gelled according to the invention intrarectally using a cannula (Colitis + Resolvix / CMC; Colitis + Resolvix / HPMC-1X; Colitis + Resolvix / HA; Colitis + Resolvix / HPMC / PC; Colitis + Resolvix / HPMC-3X). The animals were monitored daily and the clinical score quantified. The clinical score takes into account several factors cumulatively.It takes into account weight loss (0=no weight difference, 1=1-5% weight loss, 2=5-10%, 3=10-20%, 4=more than 20%), stool consistency (0=normal, 2=semi-formed and pultaceous, 4=liquid and stuck to the anus), and bleeding from the anus (0=no bleeding, 2=light bleeding, 4=presence of blood in the stool). Data are presented as cumulative data from 3 independently performed experiments, with 5 mice per group per experiment, and represented as group mean + / - standard error of the mean. At days 11, 12 and 13, all groups showed a clinical colitis score statistically different from the "Colitis + vehicle" group, except the "Resolvix / CMC" group at day 11, according to two-way analysis of variance followed by a Tukey multiple comparison test.

[0064] The results show that gelling Resolvix® according to the invention with HPMC, HA, CMC or with an HPMC / PC mixture, makes it possible to obtain an equivalent therapeutic efficacy for a quantity of Resolvix® 10 times lower compared to the quantity of Resolvix® required in its known version (1 mL vs. 100 pL). The therapeutic effect of the active ingredient Resolvix® is drastically increased by means of the polymers selected by the Applicant. Furthermore, the invention in gel form makes it possible to dispense with the intravenous route for the administration of Resolvix®. The invention thus makes it possible to administer Resolvix® locally, in a targeted manner. Very surprisingly, gelling Resolvix® with 1.5% HPMC further potentiates the therapeutic effect of Resolvix®. A synergistic effect is observed.

[0065] In summary, the results show that the invention makes it possible to increase the therapeutic activity of Résolvix®, or even to potentiate it through a synergistic effect, with a reduced quantity of the main active ingredient Résolvix®. In addition, the route of administration of Résolvix® is drastically improved and / or simplified in its form according to the invention.

[0066] The Applicant confirmed the preclinical results obtained, ex vivo by quantifying biomarkers associated with experimental colitis in the intestinal mucosa, 3 days after treatment. Thus, the weight / length ratio of the intestine, the inflammatory molecules TNF-a and MPO, and the leukocyte infiltrate (neutrophils and macrophages), at the sacrifice of the animals were studied.

[0067] Figure 5 shows plots of the reduction of inflammatory biomarkers associated with acute inflammatory colitis following different treatments using Resolvix®. Inflammatory biomarkers associated with DSS-induced acute inflammatory colitis (see Figure 4) were quantified at the end of the 13-day experiment at the colon, its weight / length ratio (Figure 5a), myeloperoxidase activity (MPO; Figure 5b), the amount of TNF-a (Figure 5c), and the number of neutrophils (Figure 5d) and macrophages (Figure 5e). Cumulative data from three independently performed experiments with 5 animals per group per experiment, represented as group mean + / - standard error of the mean. All conditions are statistically different from the "colitis" condition according to a two-way analysis of variance followed by a Tukey multiple comparison test.

[0068] The results show that the various biomarkers are reduced not only with the Resolvix® treatment used alone in intravenous administration, but also with the Resolvix® gelled treatments according to the invention (and which contains a reduced amount of supernatant). In particular, the results show that the various biomarkers are reduced with the Resolvix® treatments in its gelled form according to the invention with HPMC, HA, HMPC / PC. The embodiments of the invention with HPMC 1X and HPMC 3X are particularly effective. The results with the CMC polymer are less evident, but nevertheless satisfactory given the reduced amount of active ingredient Resolvix® in its gelled version according to the invention.

[0069] A particularly suitable gelation of the supernatant, in particular of the Résolvix® type, is obtained with the HPMC polymer. The HPMC embodiment also particularly improves the therapeutic activity in vivo. Thus, this embodiment is used in an experimental model of chronic inflammatory colitis in mice. Chronic inflammatory colitis is induced by repeated cycles of drinking water supplemented with dextran-sodium-sulfate and non-supplemented water. In this chronic model, the treatment with Résolvix® in its version alone and in its gelled version of the invention were studied. For this, Résolvix®, in its two versions, is administered at the start of the experiment (on D0), or on the tenth day (D10), by intrarectal route.

[0070] Figure 6 shows graphical representations of clinical scores of chronic inflammatory colitis, as well as mouse survival after treatment with Resolvix® gel or in its original form. 8-week-old BALB / c mice were given dextran sodium sulfate (DSS; 3% w / V) in their drinking water for 10 days and then unsupplemented drinking water for 10 days, and each cycle was repeated once. Mice received at day 10 (Figure 6a and Figure 6b) or at day 10 (Figure 6c and Figure 6d), 100 μL of Resolvix® (+ Resolvix), control solution (PBS; Colitis) or Resolvix® gelled at 0.5% and 1.5% by weight of HPMC (respectively +Resolvix / HPMC-1X and +Resolvix / HPMC-3X) intrarectally using a cannula (see black arrows in Figure 6). The clinical score (Figures 6a and 6c), as well as survival (Figures 6b and 6d) were evaluated and quantified daily (as in Figure 4), until sacrifice.Data from two representative experiments (treatments at day 0 or day 10) with 5 animals per group, and represented as group mean + / - standard error of the mean. In Figure 6a, the "+Resolvix / HPMC-1X" and "+Resolvix / HPMC-3X" conditions are statistically different from the "Colitis" condition according to a Friedman test followed by a Dunn's multiple comparisons test). In Figure 6c, the "+Resolvix" and "+Resolvix / HPMC-1X" conditions are statistically different from the "Colitis" condition according to a Friedman test followed by a Dunn's multiple comparisons test.

[0071] The results show that the form of Resolvix® gelled in HPMC at 0.5% by weight, allows a preventive therapeutic effect (figures 6a and 6b) and therapeutic (figures 6c and 6d) superior to that obtained with Resolvix® alone, not gelled. The results of the invention of Resolvix® gelled using HPMC at a content of 1.5% by weight are less satisfactory and also suggest a certain toxicity of this formulation with reduced survival in the context of therapeutic use. Thus, in a particularly preferred embodiment, the invention relates to a pharmaceutical preparation comprising a supernatant obtained from a coculture between macrophages and irradiated leukocytes (Resolvix®) comprising 0.5% by weight of hydroxylpropylmethylcellulose (HPMC).

[0072] The therapeutic effect is confirmed ex vivo by quantifying biomarkers related to inflammatory colitis. For this, the weight / length ratio of the intestine and the inflammatory molecules TNF-a and MPO are studied at the end of the experiment, when the animals are sacrificed.

[0073] Figure 7 shows diagrams of the reduction of inflammatory biomarkers associated with chronic colitis following treatments with classic Resolvix® and the version of the invention. The inflammatory biomarkers associated with DSS-induced chronic colitis (see Figure 4) were quantified at the end of the chronic inflammatory colitis model and in particular in the colon its weight / length ratio (Figure 7a), myeloperoxidase or "MPO" activity (Figure 7b) and the amount of TNF-a (Figure 7c). Data from a representative experiment with 5 animals per group, represented as group mean + / - standard error of the mean.

[0074] The data show that the effect of Resolvix® alone is at least preserved, or even improved when Resolvix® is gelled with HPMC at 0.5% and 1.5% by weight. The invention allows an improvement in the weight / length ratio of the colon of the treated animals, a reduction in MPO activity and the reduction of the tissue level of TNF-a. Particularly preferred modes for carrying out the invention have been described above: HA content preferably 0.5% by weight, CMC content preferably 0.4% by weight or PC content 0.1% by weight. However, those skilled in the art will be able to adapt the present invention on the basis of the following. In particular, for the formation of a gel suitable from the point of view of wound healing, HA can be used in a content of about 0.3 to about <1.5 wt%, while PC can be used in a content of about 0.1 to about <1 wt%.For the formation of a gel suitable from the point of view of the galenic form for rectal administration, contents allowing the obtaining of a liquid or semi-solid form are satisfactory (taking into account the risk of leakage after administration in a fluid liquid form of approximately 10 cps; or a slowing down or sequestration of the active substance for the most viscous semi-solid forms). In general, it is preferable to work in the following viscosity ranges: 15 cps < viscosity at 100s-1 < 2200 cps. Indeed, beyond 2200 cps, the results from the point of view of wound healing are no longer satisfactory. The viscosity range of a medicinal form intended for the rectal route (liquid to semi-solid) depends on both the therapeutic objective, the expected action of the active substance and the physiological context.The viscosity range for the rectal route overlaps that of the cutaneous forms, i.e. a viscosity of >10 to 100,000 cps, which corresponds to the definition of a liquid form for viscosities below 1,000 cps and semi-solid for higher values.

[0075] It is clear from the above that the present invention makes it possible to preserve the pro-resolving properties of the Résolvix® resolvetome and to increase its effectiveness. The gelled Résolvix® type supernatant according to the invention finds application in particular in the treatment of tissue inflammation of the mucous membranes such as the intestine and the skin, alone or in addition to other therapies, in human or veterinary medicine. In particular, the invention finds application in Chronic Inflammatory Bowel Diseases or "IBD". Among IBDs, mention may in particular be made of Crohn's disease (CD) and ulcerative colitis (UC). More generally, the invention is for use in the prevention and / or treatment of a pathological immune response.The use of the pharmaceutical preparation according to the invention is carried out in particular at the time of a failing resolution in order to re-establish an effective resolution and allow a halt to the inflammatory reaction.

[0076] Another subject of the invention is a pharmaceutical preparation for use in the prevention or treatment of a pathological immune response, comprising a supernatant obtained from the coculture of phagocytes with apoptotic cells, and further comprising a polymer selected from the group consisting of hydroxylpropylmethylcellulose (HPMC), hyaluronic acid (HA), carboxymethylcellulose (CMC) and polycarbophil (PC) or a mixture thereof. This embodiment relates to the combination of the Applicant's product Resolvix® with a polymer of the invention. This results in an improved version of the product Resolvix®, and this in gel form.

Claims

Claims

1. Pharmaceutical preparation comprising a supernatant obtained from a coculture between phagocytes and irradiated leukocytes, characterized in that it further comprises a polymer selected from the group consisting of hydroxylpropylmethylcellulose (HPMC), hyaluronic acid (HA), carboxymethylcellulose (CMC) and polycarbophil (PC) or a mixture thereof, wherein the weight content of the polymer is chosen so as to confer a gelled form to said pharmaceutical preparation.

2. Pharmaceutical preparation according to one of the preceding claims, in which the polymer is hydroxypropylmethylcellulose (HPMC).

3. A pharmaceutical preparation according to claim 3, wherein the hydroxylpropylmethylcellulose (HPMC) content is between 0.5% and 1.5% by weight.

4. A pharmaceutical preparation according to claim 3, wherein the hydroxylpropylmethylcellulose (HPMC) content is 0.5% by weight.

5. A pharmaceutical preparation according to claim 3, wherein the hydroxylpropylmethylcellulose (HPMC) content is 1.5% by weight.

6. Pharmaceutical preparation according to claims 1 and 2, wherein, when the polymer is hyaluronic acid (HA) its content is 0.5% by weight, when the polymer is carboxymethylcellulose (CMC) its content is 0.4% by weight, and when the polymer is polycarbophil (PC) its content is 0.1% by weight.

7. Pharmaceutical preparation according to one of the preceding claims, wherein the phagocytes are leukocytes, preferably derived from the buffy coat of whole blood.

8. Pharmaceutical preparation according to one of the preceding claims, for use as a medicament.

9. Pharmaceutical preparation according to one of the preceding claims, for use in the treatment of inflammatory diseases.

10. Pharmaceutical preparation according to one of the preceding claims, for use in the treatment of chronic inflammatory bowel disease.

11. A pharmaceutical preparation according to claim 11, wherein said chronic inflammatory bowel disease is selected from Crohn's disease (CD) and ulcerative colitis (UC).

12. Pharmaceutical preparation according to one of the preceding claims, wherein said supernatant is Resolvix®.

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