ES2914617A1 - Pharmaceutical composition for use in the treatment of inflammatory diseases (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Pharmaceutical composition for use in the treatment of inflammatory diseases. The present invention refers to a pharmaceutical composition that comprises eprinomectin. Composition is useful for the treatment of anti -inflammatory diseases, such as rheumatoid arthritis. (Machine-translation by Google Translate, not legally binding)

Description

Pharmaceutical composition for use in the treatment of inflammatory diseases

Technical Sector

The present invention relates to a pharmaceutical composition comprising eprinomectin. The composition is useful for the treatment of anti-inflammatory diseases, such as rheumatoid arthritis.

Background

Rheumatoid arthritis (RA) is a chronic autoimmune disease, characterized by inflammation in multiple joints, ultimately leading to joint deformity, pain and swelling. RA is one of the most common inflammatory arthropathies, with a prevalence of 0.3%-1%, manifesting during the most productive years of adulthood (20-40). Within 10 years of onset, at least 50% of patients are unable to hold a full-time job, and there are no therapies to prevent the disease. Therefore, RA represents a global burden and RA patients need better and safer treatments (World Health Organization, 2016; http://www.who.int/chp/topics/rheumatic/en/).

Cytokine-mediated pathways appear to be central to the pathogenic process, involving cells of the innate and adaptive immune system that infiltrate joints, as well as fibroblast-like synoviocytes (FLS). All these types of cells secrete inflammatory cytokines (such as TNF-a, IL-6, IL-1, IL-17, GM-CSF, among others), which are postulated as keys to initiate and maintain the pathogenic process, eventually leading to to joint destruction (Kojima et al. Mod Rheumatol 2020, 30(6):941-947; Smolen JS et al. Lancet 2016, 388(10055): 2023-2038).

Conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), such as methotrexate, are used as first-line therapy: they are effective in relieving symptoms but do not suppress disease progression, as they do not target the actual cause of disease. disease, altered immune responses (Chaudhari K et al. Nat Rev DrugDiscov 2016, 15(5): 305-306).

Biologic DMARDs (bDMARDs) show greater success in disease remission by targeting key immune mediators (eg, TNF-a or IL-6R) and most of them interfere with inflammatory immune pathways (Meier FM et al Immunotherapy 2013, 5(9): 955-974).

TNF inhibitors are the most widely used (in combination with methotrexate) and have been shown to be highly effective in patients who do not respond to csDMARDs; however, 20%-40% of patients still do not respond or achieve significant improvement. It is postulated that one of the causes of these inadequate responses in a high percentage of patients treated with csDMARDs and bDMARDs is because RA is not a single entity, but rather a syndrome caused by various altered pathways that operate differently in individual patients. (McInnes IB et al. Nat Rev Rheumatol 2016, 12(1): 63-68); many of these pathways may not be targeted by currently available therapies.

In this line, one of the unmet needs in RA research, highlighted both in the report of the European League Against Rheumatism (EULAR; European League Against Rheumatism Report 2017. http://www.eularcongressnewsdigital.com/eularcongressnews/ eular_2017_report?pg=1#pg1) as in the European Roadmap for Research in Rheumatic and Musculoskeletal Diseases initiative (European Roadmap for Research in Rheumatic and Musculoskeletal Diseases initiative. 2017. https://www.eular.org/public_affairs_rheumamap.cfm), is the development of new therapies for patients who do not respond to csDMARDs and/or bDMARDs, as well as the validation of new therapeutic targets. Non-adherence has also been described as a major challenge: negative feelings and distaste for methotrexate treatment have been identified as the main cause of non-adherence in 33% of patients. Regarding bDMARDs, they can only be administered parenterally: needle phobia is common and 24% of self-injecting patients experience pain on injection, leading to non-adherence (Mocsai A et al. BM CM ed 2014, 12: 43. Wright S et al. Aust Fam Physician 2009, 38(3): 172-176. McInnes IB et al. Clin Exp Rheumatol 2013, 31(3): 350-357. Schwartz DM et al. Nat Rev Rheumatol 2016, 12(1): 25-36). The World Health Organization (WHO) suggests that improving adherence would have a more beneficial effect than just improving therapeutic approaches (http://www.who.int/chp/knowledge/publications/adherence_introduction.pdf).

Therefore, the discovery of a drug capable of being administered orally would significantly improve both adherence and quality of life for the patient.

In this sense, an alternative to cs and bDMARDs are synthetic targeted DMARDs (tsDMARDs): these include small, low molecular weight molecules that, unlike bDMARDs, can target intracellular components, increasing the range of targeted pathways. treatment. tsDMARDs can be administered orally, improving compliance and thus treatment efficacy, and are cheaper to produce than bDMARDs (Mocsai A et al. BM CM ed 2014, 12:43). Therefore, they constitute a strong alternative for both csDMARD/bDMARD noncompliant and nonresponsive patients. Emerging tsDMARDs target inflammatory cytokine-activated intracellular signal transduction pathways, primarily the JAK/STAT pathway, which is downstream of type I/II cytokine receptors: e.g., JAK1/2 inhibitor /3 FDA-approved tofacitinib has shown impressive results as monotherapy, even in treatment-naïve patients unresponsive to csDMARDs/bDMARDs (Schwartz DM et al. Nat Rev Rheumatol 2016, 12(1): 25-36) .

However, these treatments do not target the actual cause of the disease: bDMARDs (for example, Anti-TNF or Anti-IL-6R) prevent the binding of the cytokine to its receptor, but the extracellular environment remains inflammatory; on the other hand, jakinibs prevent cytokine signaling in the target cell but immune cells and FLS still continue to secrete pro-inflammatory cytokines.

Thus, it is still necessary to have new DMARDs capable of reducing the inflammatory environment.

Brief description of the invention

The results presented in this document demonstrate that a pharmaceutical composition comprising eprinomectin is capable of interacting with peripheral blood mononuclear cells. More specifically, it is shown that eprinomectin is capable of reducing the levels of the proinflammatory cytokine IL-1p (example 3) and also reduces the levels of the proinflammatory cytokine TNF-a (example 4), both in cells from healthy donors as well as from patients suffering from rheumatoid arthritis and who were recently diagnosed. Furthermore, it is shown that the pharmaceutical composition comprising eprinomectin shows no toxicity (example 2).

Thus, in a first aspect the invention is directed to a pharmaceutical composition comprising eprinomectin, a pharmaceutically acceptable salt thereof, or an analog thereof, for use in the treatment of an inflammatory disease.

In a particular embodiment, the inflammatory disease is rheumatoid arthritis.

In another particular embodiment, eprinomectin, a pharmaceutically acceptable salt thereof or an analog thereof, in the composition of the invention, is found in combination with an additional active ingredient. In a more particular embodiment, said additional active ingredient is an anti-inflammatory drug or cytokine antagonist.

In another particular embodiment, the pharmaceutical composition of the invention is administered in a pharmaceutical form suitable for oral, intravenous, subcutaneous or intramuscular administration.

In another particular embodiment, the pharmaceutical composition of the invention is in liquid or lyophilized form.

In a second aspect, the invention is directed to a method of treating an inflammatory disease, characterized by administering to an individual in need a therapeutically effective amount of the pharmaceutical composition described in the first aspect of the invention. In a particular embodiment, the inflammatory disease is rheumatoid arthritis.

In another particular embodiment, an additional active ingredient is administered to the individual. In a more particular embodiment, said additional active ingredient is an anti-inflammatory drug or cytokine antagonist.

Description of the figures

Figure 1 depicts the cell viability of 6 DS PBMCs inflamed in vitro and subsequently treated with 7 different concentrations of eprinomectin or vehicle. The viability index is shown, calculated as the ratio between the luminescence value of the compound and that of the vehicle, at each concentration. The median (circles) and interquartile range are shown for each concentration tested.

Figure 2 represents the ratios of the values obtained for the amount of IL-ip secreted by the in vitro model of inflammation in supernatants of cells treated with eprinomectin or vehicle versus untreated cells. Values below 1 indicate reduced cytokine secretion. Circles and solid lines: eprinomectin. Squares and broken lines: vehicle. Paired Student's t-test. **: p<0.01.

Figure 3: Figure 3A represents, for donor DS_008, the amount of IL-i p (in pg/mL) secreted by the in vitro model of inflammation treated with eprinomectin (solid lines) or vehicle (dashed lines). Figure 3B depicts the same for donor DS_009. Figure 3C depicts the same for donor DS_013. Figure 3D represents the same for donor DS_014. Figure 3E depicts the same for donor DS_016.

Figure 4: Figure 4A represents the eprinomectin/vehicle ratio (%) of the percentage of PBMCs expressing IL-i p (“IL-1- P + PBMCs”): positive values indicate increased frequency of IL-1- P + PBMCs after treatment of the in vitro model of inflammation with eprinomectin; negative values denote reduction. Results are shown for 3 naive patients (N) and 4 healthy donors (DS). Figure 4B represents the equivalent of Figure 4A for CD14neg IL-1- P + monocytes. *: p<0.05. Student's paired t-test.

Figure 5 represents that the addition of PHA-L to our model improves the detection and quantification window of TNF -a . Student's t-test. ***: p<0.001. LPS ATP: in vitro model of inflammation. LPS ATP PHA: modified in vitro model of inflammation.

Figure 6: Figure 6A represents the amount of TNF - α produced after treatment with vehicle or eprinomectin by the modified in vitro model of inflammation. Circles: vehicle. Squares: eprinomectin. Open symbols: patients. Closed symbols: DS. Wilcoxon rank test. *: p<0.05. Figure 6B represents the vehicle/eprinomectin ratios (in %) of the data shown in Figure 6A. Positive values indicate increased secretion of TNF - α after treatment with eprinomectin of the modified in vitro model of inflammation; negative values denote reduction. Wilcoxon rank test. *: p<0.05.

Figure 7: Figure 7A represents the eprinomectin/vehicle ratio (%) of the percentage of PBMCs expressing TNF - α (“PBMCs TNF - α +”): positive values indicate increased frequency of TNF-a+ PBMCs after eprinomectin treatment of the modified in vitro model of inflammation; negative values denote reduction. Results are shown for 3 naive patients (N) and 4 healthy donors (DS). Figure 7B represents the equivalent of Figure 7A for CD14neg TNF-a+ monocytes. Figure 7C represents the equivalent of Figure 7A for TNF-a+ lymphocytes. *: p<0.05. Student's paired t-test.

Figure 8: Figure 8A represents the eprinomectin/vehicle ratio (%) of the mean fluorescence intensity of TNF in TNF-a+ PBMCs: positive values indicate higher amount of TNF in TNF-a+ PBMCs after treatment with eprinomectin of the in vitro model of modified inflammation; negative values denote reduction. Results are shown for 3 naive patients (N) and 4 healthy donors (DS). Figure 8B represents the equivalent of Figure 8A for TNF-a+ lymphocytes. *: p<0.05. Student's paired t-test.

Detailed description of the invention

Eprinomectin is a semi-synthetic compound of the avermectin family, used for the treatment of internal and external parasites in cattle, including lactating cows. In the present invention, eprinomectin includes a mixture of two homologues, eprinomectin B1a and eprinomectin B1b, which differ by one methylene group at C25, and also includes each of the homologues separately. The percentage of the homologues in the mixture varies from 99/1 to 1/99, for example they are in a 90/10 or 10/90 ratio. The mode of action of eprinomectin is unknown, despite a large number of studies carried out on a variety of compounds in the same class (European Medicines Agency, (https://www.ema.europa.eu/en /documents/mrl-report/eprinomectin-summary-report-1-committee-veterinary-medicinal-products_en.pdf).

By "peripheral blood mononuclear cells or PBMCs" is meant cells with a single nucleus isolated from human blood by density gradient, and which are components of the immune system (lymphocytes, monocytes and dendritic cells). These cells are key components of the immune system, being easily accessible and manipulable, which is why they have been selected for this invention.

"IL-1p proinflammatory cytokine" is understood as the protein interleukin 1 beta, produced by cells of the immune system and by other cell types, and which causes the propagation of the inflammation. The levels of proinflammatory cytokine IL-ip are elevated in inflammatory processes, such as rheumatoid arthritis, constituting one of the causal agents of the etiopathogenesis of said disease.

It is understood by "reducing the levels of the proinflammatory cytokine IL-ip" to the reduction of the amount of the proinflammatory cytokine IL-1p produced by cells of the immune system and/or fibroblasts in the extracellular medium in a subject suffering from a process or pathology inflammatory.

The term "proinflammatory cytokine TNF-a" is understood as the protein tumor necrosis factor alpha, produced by cells of the immune system and by other cell types, and which causes the spread of inflammation. Levels of the proinflammatory cytokine TNF-a are elevated in inflammatory processes, such as rheumatoid arthritis, constituting one of the causal agents of the etiopathogenesis of said disease.

It is understood by "reducing the levels of the proinflammatory cytokine TNF-a" to the reduction of the amount of the proinflammatory cytokine TNF-a produced by cells of the immune system and/or fibroblasts in the extracellular medium in a subject suffering from a process or pathology inflammatory.

" In vitro model of inflammation" is understood as PBMCs stimulated with Escherichia coli lipopolysaccharides (LPS) for 3h and adenosine triphosphate (ATP) for 40', and washed with culture medium to remove said compounds, before treatment with vehicle or eprinomectin.

"Modified in vitro model of inflammation" is understood as the previously described in vitro model of inflammation, to which phytohemagglutinin L (PHA-L) is added after removal by washing of LPS and ATP, and before treatment with vehicle or eprinomectin. .

“Healthy donors” (“SD”) are understood as individuals between the ages of 35 and 60, with no family history of immune-mediated chronic inflammatory disease or autoimmune diseases.

"Inflammatory disease" is understood as a disease mediated by inflammatory proteins and cellular pathways, and which is modified by anti-inflammatory treatments.

Rheumatoid arthritis is an autoimmune disease that causes pain, swelling, and stiffness in the joints, and can seriously damage them. In addition, it sometimes causes loss of function and disability.

By "patients" ("N") are meant individuals suffering from rheumatoid arthritis. In this case we are referring to patients newly diagnosed with rheumatoid arthritis who have not yet received any treatment (“naive”).

In any event, the compositions of the invention may be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, that is, the material can be administered to a subject, together with the active ingredient, without causing any undesirable biological effects or interacting in a deleterious manner with any of the the other components of the pharmaceutical composition in which it is contained. The carrier may be selected to minimize any degradation of the active ingredient, to minimize any adverse side effects, and/or to direct the active ingredient to a target or tissue, as would be well known to those skilled in the art.

The effective dosage and schedules for administration of the composition of the invention can be determined empirically, and making such determinations is within the skill of the art. Dosage ranges for administration of the compositions are those large enough to produce the desired effect on the pathology. The dose should not be so large as to cause adverse side effects. Generally, the dosage will vary with age, sex, extent of disease in the patient, route of administration, or whether other active ingredients are included in the dosage and administration regimen, which can be determined by a person skilled in the art. matter.

The following examples merely serve to illustrate the invention.

Example 1: In vitro model of inflammation in primary cells of the immune system.

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by density gradient centrifugation using the commercial product Lymphoprep (Stemcell Technologies, ref#07811), following manufacturer's instructions. PBMCs were counted and resuspended in human AB serum supplemented with 20% dimethylsulfoxide (DMSO), at a concentration of 10-20x106 cells/mL. Cells were kept at -80C for 24 h, and then stored in liquid nitrogen until use.

For each of the experiments described below, cells were thawed and swollen in vitro as follows: the vial of cells was removed from liquid nitrogen and immersed in a 37oC bath until almost completely thawed. At that time, 2 mL of culture medium was added to the vial (RPMI 1640 commercial medium supplemented with penicillin, streptomycin, and 10% human serum), the content was transferred to a centrifuge tube, and culture medium was added up to 10 mL. After centrifugation at 300xg for 10 min, the supernatant was removed and the washing process was repeated. Cells were counted and resuspended at a concentration of 4x106 cells/mL. For each treatment, 2x106 cells were cultured in medium containing Escherichia Coli lipopolysaccharides (Sigma-Aldrich, ref #L4391-1MG, final concentration of 5^,g/mL), for 3h at 37°C 5% CO ² . After this time, ATP (Sigma-Aldrich, ref #A6419, final concentration of 5mM) is added, and it is incubated again at 37°C 5% CO ² for 40 minutes.

For quantitative PCR, cells were treated with LPS and ATP, washed, and lysed with RLT buffer (QIAGEN, kit component ref # 74104). The lysate was stored at -80C until use. RNA was isolated using the RNeasy Mini kit (QIAGEN, ref # 74104), following the manufacturer's instructions. RNA was eluted twice with 30^L DEPC-treated water (Invitrogen, ref # AM9915G), and quantified with a NanoDrop 1000 spectrophotometer (Thermo Scientific). The RNA was used in its entirety for the synthesis of complementary DNA, using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ref #4368814). For quantitative PCR (qPCR), the 2x Fast SYBR Green Master Mix (ThermoFisher, ref #4385610) was used, following the manufacturer's instructions, in a QuantStudio® 12K Flex Real-Time PCR System, 96-well block, desktop (Life Technologies , Singapore). For the analysis of the results, the standard method of AACt (Livak, KJ and TD Schmittgen. Methods 2001. 25 (4): 402-408) was used. For analysis by flow cytometry, brefeldin A (BioLegend, ref #420601) was added at 5^g/mL simultaneously to LPS: Brefeldin A is a macrolide antibiotic used to enhance intracellular cytokine staining signals thanks to its ability to blocking of transport processes during cell activation, thus retaining the cytokines inside the cell and allowing their detection by flow cytometry.

After incubation with brefeldin A, LPS and ATP, the cells were harvested and washed with culture medium at 400xg 10'. During centrifugation, 1mL of cold PBS is added to make it easier for the cells adhered to the bottom to detach. The supernatant is then discarded and the cells are resuspended with PBS. It is centrifuged again at 400xg 10', subsequently discarding the supernatant. The cells are fixed and permeabilized by incubation with the commercial compound Cytofix/Cytoperm (BD Biosciences, ref #554722) in the cold for 20' at 4°C. Next, 1 ml of cold commercial Perm/Wash 1X buffer (BD Biosciences, ref #554723) is added and centrifuged at 400xg 10'. The supernatant is discarded and the wash is repeated. The supernatant is then removed trying to leave very little volume.

Cells are stained with the corresponding primary antibody (Anti-NLRP3 or isotype, Abcam, ref #ab4207 and #ab37373. Final concentrations of 10^g/mL), overnight at 4oC in the dark. Two washes are then performed as described above, and cells are stained with a PE-conjugated secondary antibody specific for Anti-NLRP3 and isotype (Abcam, ref # ab7004, final concentration 3^g/mL) , and/or Anti-IL6/FITC antibody (Biorbyt, ref #orb102822, final 1^1/50^1), and/or Anti-TNF-a/AF647 (Biolegend, ref #502916 2.5^L/50 ^L end). After two washes the cells are resuspended in 400^L of Perm/Wash buffer and analyzed in a FACSCalibur (BD) cytometer. The results are analyzed using the FlowJo software (BD).

Cytokine quantification is usually carried out by ELISA ( Enzyme-LinkedImmunoSorbent Assay) assays. However, an assay has to be performed for each cytokine of interest, decreasing the efficiency of the process. In this case we have chosen to use a multiplex cytokine quantification technique, which allows the quantification of 10 different cytokines using only 50 |iL of sample. This technique is based on the use of magnetic beads labeled with different fluorophores and with specific antibodies for different cytokines. We quantified 10 cytokines (GM-CSF, IFN-y, IL-1P, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-a. ThermoFisher Scientific, ref #LHC0001M ) in culture media. The luminescence reading took place in a Luminex XMAP reader (Bio-Rad). For the analysis of the results, the manufacturer's instructions were followed: the values of mean fluorescence intensity (MFI) and the known concentrations of the standards for each cytokine to develop standard curves using a 5-parameter logistic model. The concentration values of each sample were then extrapolated by entering the corresponding MFI values for each cytokine and curve. In case the MFI values were above the curve, the raw MFI value is shown instead of the extrapolated concentration value.

Thus, we developed an in vitro model of inflammation, consisting of the sequential stimulation of peripheral blood mononuclear cells (PBMCs) from healthy donors with Escherichia coli lipopolysaccharides (LPS) and adenosine triphosphate (ATP).

One of the key components in the cellular inflammatory process is the NLRP3 inflammasome: the NLRP3 protein is a member of the nucleotide binding domain (NOD) receptor family whose expression can be found in the cytoplasm of a wide variety of cells, both from the immune and non-immune systems, but having a particular importance in cells of the innate immune system (Ahn, H. et al. J Biomed Res 2018. 32(5): 401-410). After its activation, the NLRP3 protein associates with an adapter protein ( apoptosis-associated specklike protein, ASC) and an effector protein (pro-caspase-1), constituting the NLRP3 inflammasome (Shen, HH et al, Autoimmun Rev 2018. 17( 7): 694-702). This protein complex promotes inflammation through the processing of IL-1P and IL-18 from their inactive forms to their active forms, which in turn, when secreted, promote the activation of other pro-inflammatory pathways. Activation of the NLRP3 sensor requires 2 signals: a first stimulatory signal through pattern recognition receptors (PRR) that causes increased expression of NLRP3, and a second activating signal that induces inflammasome oligomerization (Shen, HH et al, Autoimmun Rev 2018. 17(7): 694-702). Therefore, we decided to base our model on the sequential stimulation of peripheral blood mononuclear cells (PBMCs) from healthy donors with lipopolysaccharides from Escherichia coli (LPS, a PRR ligand that acts as the first signal) and adenosine triphosphate (ATP, which acts as second signal). This type of sequential stimulation has previously been used successfully to induce inflammation in other studies (e.g. Choulaki, C. et al, Arthritis Res Ther, 2015. 17(1): 257, and Mouton-Liger, F. et al . al, Glia 2018. 66(8): 1736-1751).

To verify that, indeed, the in vitro model of inflammation is valid, a quantitative PCR (qPCR) was performed, which showed that after the culture of PBMCs with LPS there is an increase in the expression of NLRP3, and also an increase in the expression of IL-1fi. These results were confirmed at the protein level by flow cytometry. Also, after the Treatment with LPS and ATP shows a clear increase in the percentage of IL-1P+ cells, mainly due to the production of IL-1p by CD14+ monocytes.

In addition, LPS is considered one of the most powerful stimuli for the activation of the innate immune system, inducing the production of a myriad of proinflammatory cytokines, such as TNF-a, IL-6, IL-8, the IL-12 family, other IL-ip family cytokines, IL-15 and TGF-P (Rossol, M. et al, CritRevImmunol 2011. 31 (5): 379-446) To confirm that this is true in our model, we carried out a qPCR, confirming that treatment with LPS causes increased expression of TNF-a and IL-6. We also checked the presence of the proteins by flow cytometry, confirming that both IL-6 and TNF-a are expressed in PBMCs treated with LPS and ATP.

Finally, we studied whether proinflammatory cytokines are secreted into the extracellular medium, since this is how the inflammatory process would propagate physiologically. For this, we treated PBMCs from 6 healthy donors (DS) with LPS and ATP, and analyzed the presence of 10 cytokines in multiplex in the culture medium. Treatment with LPS and ATP induces a significant increase in the secretion of IL-ip, IL-6 and GM-CSF, all of which are proinflammatory. We also observed an increase in the secretion of other proinflammatory cytokines such as TNF-a and IFN-y, although they did not reach statistical significance. A significant increase in IL-2 and IL-10 is also observed: although this last cytokine is anti-inflammatory, it has been previously described that treatment with LPS induces its secretion, possibly to balance the inflammatory response (Rossol, M. et al , Crit Rev Immunol 2011. 31 (5): 379-446).

Therefore, our in vitro model of inflammation, based on the sequential stimulation of PBMCs with LPS and ATP, leads to the activation of multiple inflammatory pathways, and is suitable for identifying new compounds capable of reducing the secretion of proinflammatory cytokines.

Example 2: toxicity

In this example we study the toxicity of eprinomectin in our in vitro model of inflammation, to establish the range of concentrations at which the compound does not cause cell death, and rule out that the results are due to the interference of dead cells.

First, we dissolved commercial eprinomectin (MolPort, ref # MP-0000-0000-3812-289) in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich, ref #D2650), at a final concentration of 10"2M (stock). Starting from this stock solution, we prepared a 10"3M solution of eprinomectin in culture medium without serum, this being the working solution that we used in all subsequent experiments. Both the stock and the working solution were kept at -20oC.

We then treated our in vitro model of inflammation with eprinomectin at two concentrations (10-5 M and 10"7 M): we decided not to exceed the concentration of 10"5 M since higher concentrations would mean a greater addition of DMSO, which at High concentrations could cause cell damage. Eprinomectin dilutions were prepared by diluting the working solution (10 -3 M) in serum-free medium to 2x concentration and added to cells, resuspended in serum-containing medium.

Cell viability is assessed by measuring ATP, since the concentration of this molecule decreases significantly in necrotic or apoptotic cells. Cell viability was quantified using the ATPlite Luminescence Assay System (PerkinElmer, ref #6016943), following the manufacturer's instructions, 21h and 46h after adding the compound.

As can be seen in Table 1, eprinomectin is not toxic at either of the two concentrations tested, both at 21h and 46h.

To confirm these results and confirm that they can be extrapolated, we repeated the viability assay in cells from 6 healthy donors using a greater range of eprinomectin concentrations, and quantifying viability after 46 h of culture. Indeed, eprinomectin is not toxic in most of the concentrations tested, with the only exception of the highest concentration, 3x10"5M, in which case the viability is reduced to approximately half, compared to the vehicle (Figure 1 and Table 2). ).

Therefore, eprinomectin is not toxic at concentrations below 3x105M.

Table 1: Luminescence values obtained in the toxicity test.

Table 2: Viability indices obtained in the toxicity assay shown in Figure 1, for each healthy donor (DS) and treatment. The indices were calculated by dividing the value obtained for eprinomectin with respect to the value obtained for the vehicle. Values less than 1 indicate decreased viability, compared to the vehicle, after treatment with eprinomectin, na: not analyzed.

Example 3: Study of the effect of treatment with eprinomectin on the production capacity and secretion of IL-ip.

To study whether the treatment of our in vitro model of inflammation with eprinomectin reduces the secretion of IL-1p, the culture media from Example 1 were thawed, centrifuged, and the cytokines quantified in multiplex, as described in the Example 1.

For the detection and quantification of intracellular IL-ip, PBMCs from 4 DS and 3 patients were incubated with LPS and ATP as in Example 1, and treated with 10 -5M eprinomectin for 48h. After 43h of culture, brefeldin A (5^g/mL) was added to maintain the cytokines inside the cell. After 48h, the cells were centrifuged, the culture media were frozen at -80C, and the cells were stained for analysis by flow cytometry.

Briefly, cells were washed twice with cold PBS, and stained with LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation, a reagent that only stains cells. apoptotic, necrotic or dying, according to the manufacturer's instructions (Invitrogen, ref # L34965 ). After two washes with FACS buffer (PBS supplemented with 5% fetal bovine serum), cells were stained with a FITC-conjugated Anti-CD14 monoclonal antibody (eBiosciences, ref #11-0149-42. 1^,g/sample) in 100^L of FACS buffer, for 30' on ice and in the dark. Cells were then washed twice with FACS buffer, fixed and permeabilized with 150^L Cytofix/Cytoperm. After two washes with Perm/Wash buffer, cells were stained with a PE-conjugated Anti-IL-1p monoclonal antibody (BD, ref #340516. 3^g/sample) in 100^L of Perm/Wash buffer, for 30 ' in the dark, after which they were washed twice and stored at 4oC until their acquisition in a CSampler cytometer (Accuri) the following morning. As controls for compensation, compensation beads ( Anti-mouse Ig, k, comp beads, BD, ref #552843) were stained with Anti-CD14/FITC or Anti-IL-1p/PE. To have a staining control for Aqua, from a sample of 1x106 PBMCs, 1x106 cells were taken and lysed by incubation for 10' at 56oC, after which they were reincorporated into the tube and stained only with Aqua. A sample of cells was also left unstained.

The analysis of the results was carried out using the FlowJo software: the compensation was carried out with the tubes stained for each individual fluorochrome. Live cells (negative for Aqua) were selected and subdivided, based on forward scatter (FSC), side scatter (SSC) and CD14 expression parameters, into total PBMCs (“PBMCs”), lymphocytes, CD14+ monocytes and CD14neg monocytes. To obtain a normalized response for all DS and patients, both in the case of the percentages of IL-1P+ cells and their mean fluorescence intensity for IL-13, the ratio “value with eprinomectin/value with vehicle” was calculated. Then, using the formula “(ratio*100)-100”, the reduction percentage was obtained: negative values indicate a reduction in the variable of interest when treating with eprinomectin compared to treatment with vehicle alone; positive values are indicative of an increase. To statistically compare the results in cells treated with eprinomectin versus those treated only with vehicle, the Prism software was used: whether or not the data followed a normal distribution was analyzed: if so, the Student's paired t-test was used; Otherwise, the Wilcoxon rank test was used.

After confirming the effect of eprinomectin on cell viability, we proceeded to verify whether this compound has the capacity to reduce the secretion of the proinflammatory cytokine IL-1p. For this we treat 5 DS PBMCs with LPS and ATP, to then incubate them with different eprinomectin concentrations for 48h. After that time, the cytokines secreted in the culture medium were qualified by Luminex, as in Example 1.

Eprinomectin reduces IL-ip secretion (eprinomectin/medium ratios below 1.0), this reduction being more apparent at the 10"5M concentration (Figure 2). This reduction is not attributable to vehicle, since the values with eprinomectin are significantly lower than vehicle alone (Figure 2, compare solid and dashed line. p=0.0035. Student's paired t-test) Therefore, eprinomectin causes the reduction in IL-ip secretion in our model in vitro of inflammation, and said reduction is not attributable to the vehicle. This reduction at 10"5M is observed in all donors analyzed (Figure 3 and Table 3), and the mean percentage reduction at said concentration is 26.93% ± 6.84 %.

Table 3: Values used in Figure 2. Ratios of the values obtained for IL-ip in supernatants of cells treated with eprinomectin or vehicle versus untreated cells. Values below 1 indicate a reduction in the secretion of the cytokine by eprinomectin.

Once we observed that the treatment of our in vitro model of inflammation with eprinomectin reduces the secretion of the proinflammatory cytokine IL-ip, we proceeded to check whether this effect is also observed in the levels of intracellular cytokine, in addition to that secreted into the medium of crop. In this case, we not only use PBMCs from healthy donors, but also cells from patients recently diagnosed with rheumatoid arthritis and who have not yet received any treatment (“naive”), so their immune response has not yet been altered by drugs.

To do this, after centrifugation and storage of the culture media, the cells were stained for different markers and analyzed by flow cytometry (see Methodology). Briefly, cells were stained with Anti-CD14/FITC, a marker that allows distinction between living cells and apoptotic/necrotic/dying cells ( Aqua, to confirm that anti-cytokine antibodies are not binding to non-viable cells), and Anti-IL-ip/PE.

The percentage of IL-ip+ cells increases after treatment with eprinomectin compared to vehicle, this increase being significant in PBMCs (Figure 4A) and CD14neg monocytes (Figure 4B). Therefore, eprinomectin reduces the secretion of IL-ip into the culture medium by preventing either the secretion or processing of IL-ip, which is reflected by an increase in the percentage of cells containing IL-ip. This effect is most apparent on CD14neg monocytes.

Therefore, eprinomectin reduces the secretion of IL-ip, both in healthy donors and in patients with rheumatoid arthritis.

Table 4: Percentages of reduction in the percentage of IL-ip+ cells and in their average fluorescence intensity, represented in Figure 4.

Example 4: Study of the effect of treatment with eprinomectin on the production and secretion capacity of TNF-a.

To increase the amount of TNF-α secreted into the medium and improve our detection window for this cytokine, we developed a modified in vitro model of inflammation, in which cells were treated with LPS and ATP as in Example i, and after two washed were incubated with PHA-L (eBioscience, ref #00-4977-93) and with eprinomectin or vehicle for 48h. After 43h, brefeldin A was added, and at 48h the cells were centrifuged and stained as indicated below; culture media were frozen at -80oC.

For TNF-a quantification in this modified model, culture media were thawed, centrifuged, and TNF-a quantified using the AlphaLISA Human TNF-a Biotin-Free Detection Kit (Perkin-Elmer, ref # AL325), following instructions. manufacturer. The plate was read on an Envision reader. During the analysis, to obtain a normalized response for all samples, the ratio "value with eprinomectin/value with vehicle" was calculated. Then, using the formula "(ratio*100)-100", the reduction percentage was obtained: negative values indicate a reduction in TNF-a secretion when treated with eprinomectin compared to treatment with vehicle alone; positive values are indicative of an increase. To statistically compare the results in cells treated with eprinomectin versus those treated only with vehicle, the Prism software was used: it was analyzed whether the data (pg/mL TNF-a) followed a normal distribution or not: if so, the t test was used. Student's pairing; Otherwise, the Wilcoxon rank test was used.

Regarding the detection of intracellular TNF-a by flow cytometry, the same protocol was followed as described in Example 3, with the only modification of reagents used in the staining: Red fluorescent reactive dye (Invitrogen , ref # L34971) instead of Aqua, and an Anti-TNF-a monoclonal antibody conjugated to AlexaFluor647 (BioLegend, ref # 502916. 5^L/sample) instead of an Anti-IL1p. Samples were acquired on a FACSCalibur cytometer. As controls for compensation, compensation beads ( Anti-mouse Ig, k, comp beads, BD) were stained with Anti-CD14/FITC or Anti-TNF-a/AF647. To have a staining control for Red fluorescent reactive dye, from a sample of 1x106 PBMCs, 1x106 cells were taken and lysed by incubation for 10' at 56C, after which they were reincorporated into the tube and stained only with Red fluorescent reactive dye. . A sample of cells was also left unstained. The analysis of the results was carried out in the same way as in Example 3.

After confirming the effect of eprinomectin on cell viability, we proceeded to verify whether this compound has the capacity to reduce the secretion of the proinflammatory cytokine TNF-a. To increase the amount of TNF-a secreted by our in vitro model of inflammation, and thus improve our detection window, we added phytohemagglutinin-L (PHA-L) to the culture medium (see methodology): this mixture of lectins is used to in vitro activation of leukocytes, and its use increases cytokine secretion (Ceuppens, JL et al , JImmunol 1988.

141 (11): 3868-3874). As can be seen in Figure 5, the addition of PHA-L causes a significant increase in the production of TNF-a (p=0.0001. Student's t-test), increasing our quantification window.

Once the model was modified, we treated the cells with 10"5M eprinomectin and quantified TNF-a using a high-sensitivity ELISA assay (AlphaLISA). In this case, we used PBMCs from healthy donors, and also from newly diagnosed patients with rheumatoid arthritis and who have not yet received any treatment (“naive”, N in the figures and tables), so their immune response has not yet been altered by drugs.

Indeed, eprinomectin induces a significant reduction in TNF-a secretion in both DS and patients (Figure 6A,B. p=0.0156. Wilcoxon rank test). The mean reduction (relative to vehicle) is 33.4% ± 21.6%, and only in one donor (N_005) eprinomectin does not show this reducing effect.

Having confirmed that treatment with eprinomectin in our modified in vitro model of inflammation reduces the secretion of the proinflammatory cytokine TNF-a, we proceeded to check whether this effect could also be detected at the intracellular level.

To do this, after centrifugation and storage of the culture media, the cells were stained for different markers and analyzed by flow cytometry. Briefly, cells were stained with Anti-CD14, a marker that allows distinction between living cells and apoptotic/necrotic/dying cells (to confirm that anti-cytokine antibodies are not binding non-viable cells), and Anti-TNF. -a.

The percentage of TNF-a+ cells increases after treatment with eprinomectin, this increase being significant in PBMCs, and CD14neg monocytes, almost also reaching statistical significance in lymphocytes (Figure 7). These results are identical to those obtained by quantification of TNF-α in the extracellular medium (compare Figures 6B and 7A).

The amount of cytokine present in TNF-a+ cells is significantly lower in PBMCs and lymphocytes after eprinomectin treatment, quantified as mean fluorescence intensity (MFI) (FIG. 8). Therefore, eprinomectin reduces both the frequency of TNF-a+ cells and the amount of TNF-a present in said cells, the latter effect being lymphocyte-specific.

Therefore, eprinomectin reduces the secretion of TNF-a, both in healthy donors and in patients with rheumatoid arthritis.

Table 5: Percentages of reduction in the percentage of TNF-a+ cells and in their average fluorescence intensity, represented in Figures 7 and 8.

Claims (5)

1. A pharmaceutical composition comprising eprinomectin, a pharmaceutically acceptable salt thereof, or an analog thereof, for use in the treatment of an inflammatory disease. 2. Pharmaceutical composition according to claim 1, wherein the inflammatory disease is rheumatoid arthritis. 3. Pharmaceutical composition according to any of the preceding claims, wherein eprinomectin, a pharmaceutically acceptable salt thereof or an analog thereof is in combination with an additional active ingredient. 4. Pharmaceutical composition according to any of the preceding claims, wherein said composition is administered in a pharmaceutical form suitable for oral, intravenous, subcutaneous or intramuscular administration. 5. Pharmaceutical composition according to any of the preceding claims, wherein said composition is in liquid or lyophilized form.