REDUCTION OF BONE RESORPTION, ESPECIALLY IN CHRONIC JOINT DISEASES

Abstract

A TLR7/8 inhibitor for reduction of bone resorption, especially in chronic joint diseases, and to a pharmaceutical composition including the inhibitor. A method for predicting the severity of the course of disease of rheumatoid arthritis in a patient.

Claims

1. A method comprising the step of administering a TLR7/8 inhibitor for the reduction of bone resorption in the case of diseases which are accompanied by an excessive bone resorption, in particularly in the case of diseases which are selected from the group consisting of chronic joint diseases, acute joint diseases and periodontitis, and/or in the case of disorders of the engraftment of implants.

2. The method according to claim 1, wherein the disease is a chronic joint disease which is selected from the group consisting of rheumatoid arthritis and arthrosis.

3. The method according to claim 1, wherein the TLR7/8 inhibitor is a direct or an indirect inhibitor, wherein a direct TLR7/8 inhibitor is a component which interacts with TLR7/8 in a direct physical manner and thus results in an inhibition of TLR7/8, and wherein an indirect TLR7/8 inhibitor is a component which results in an inhibition of TLR7/8 by inhibiting one r more TLR7/8 agonists and/or activating one or more TLR7/8 antagonists.

4. The method according to claim 1, wherein the inhibitor is selected from the group consisting of miR-574-5p-AntagomiR, miR574-5p-sponge, miR574-5p-decoy, hydroxychloroquine, hydroxychloroquine sulfate, chloroquine, quinacrine (=mepacrine), CpG-52634, SM934, ST2825, IRS-661, IRS-954, DV-1179, IMO-3100, IMO-8400, IMO-9200, IHN-ODN-24888 and ODN 2087 (ODN 2088 Control (ODN2087)).

5. The method according to claim 1, wherein the inhibitor is a single stranded RNA analog which is complementary to miR-574-5p (SEQ ID NO: 4) (AntagomiR).

6. The method according to claim 5, wherein the inhibitor is a PNA AntagomiR.

7. The method according to claim 1, wherein the bone resorption is reduced by means of an inhibition of the osteoclast differentiation.

8. A pharmaceutical composition comprising a TLR7/8 inhibitor according to claim 1.

9. The pharmaceutical composition according to claim 8, wherein the composition contains carriers which are selected from the group consisting of nanocarriers (NCs) and/or cholesterol.

10. The pharmaceutical composition according to claim 9, wherein the NCs are nanoparticles (NPs).

11. The pharmaceutical composition according to claim 9, wherein the carriers are iron oxide nanoparticles.

12. The pharmaceutical composition according to claim 10, wherein the nanoparticles have a diameter in a range of 5 nm to 100 nm.

13. The pharmaceutical composition according to claim 9, wherein the inhibitor is present covalently bound to the carrier.

14. The pharmaceutical composition according to claim 8, wherein the composition contains carriers which are cell penetrating peptides (CPPB).

15. The pharmaceutical composition according to claim 14, wherein the TLR7/8 inhibitor is covalently bound to the CPP by means of a PEG linker.

16. The pharmaceutical composition according to claim 14, wherein the TLR7/8 inhibitor is a single stranded RNA analog which is complementary to miR-574-5p (SEQ ID NO: 4) (AntagomiR).

17. A method for the prognosis of the severity of the course of the disease rheumatoid arthritis of a patient comprising the following steps: a) determining the content of miR-574-5p in sEVs (small extracellular vesicles) in a sample of the patient, b) comparing the content determined in step a) with the content of miR-574-5p in sEVs in samples of one or more comparison patients with a known course of the disease, and c) prognosticating the severity of the course of the disease based on the result of the comparison.

18. The method according to claim 17, wherein the content of miR-574-5p is determined with RT-qPCR.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0050] FIG. 1 shows schematically the isolation of sEVs with differential ultracentrifugation.

[0051] FIG. 2 shows schematically the induction of osteoclast genesis.

[0052] FIG. 3 shows a significant concentration-dependent increase of the osteoclasts, when sEVs are added to monocytes (FIG. 3A) or to M2-like macrophages (FIG. 3B). The results are indicated as mean value+SEM (standard error of the mean). Differences were classified as significant for p<0.05 (indicated as * for p<0.05, ** for p<0.01, *** for p<0.001).

[0053] FIG. 4 shows the content of different miRs in the isolated vesicles determined with the help of RT-qPCR. A high content of miR-574-5p (SEQ ID NO: 4) was ascertained in both the sEVs from the serum and the sEVs from the synovial fluid (FIGS. 4A and 4B). The results are indicated as mean value+SEM.

[0054] FIG. 5 shows intracellular levels and sEV levels of the indicated miRs with and without stimulation for 24 hours with IL-1β (10 ng/ml) and/or TNFα (10 ng/ml).

[0055] FIG. 6 shows intracellular levels (FIG. 6A) and secretion (FIG. 613) of miR-574-5p during the osteoclast differentiation.

[0056] FIG. 7A shows the content of miR-574-5p in miR-574-5p oe sEVs and in a control (ScrC sEVs). FIG. 7B shows results of an RNase protection experiment, whereby the RNase degradation of the miR-574-5p can significantly be increased by an addition of a detergent.

[0057] FIG. 8 shows the results of a treatment with 1 μg/ml miR-574-5p oe sEVs or ScrC sEVs at different time points of the differentiation. The miR-574-5p oe sEVs lead to a significant increase of the osteoclast number, when added to monocytes or M2-like macrophages (FIGS. 8A and 8B). In the case of the addition to pre-osteoclasts, on the other hand, such a significant increase of the osteoclast number was not observed (FIG. 8C).

[0058] FIG. 9 shows that neither miR-574-5p alone, nor miR-574-5p in the presence of synthetic liposomal vehicles (Lipofectamine® 2000) results in a stimulation of the osteoclast genesis.

[0059] FIG. 10 shows a direct interaction of miR-574-5p with TLR8 by means of MST (microscale thermophoresis). The constant of dissociation K.sub.D was 30.8±5.2 nM (FIGS. 10A and 10B). However, a specific interaction between TLR8 and miR-16-5p was not observed (FIGS. 10A and 10B). FIGS. 10C and 10D show that an addition of the TLR7/8 inhibitor ODN 2087 nullifies the effect of miR-574-5p onto CD14.sup.+ monocytes and M2-like macrophages. FIGS. 10E and 10F show the influence of the TLR7/8 ligand R848 onto the osteoclast genesis.

[0060] FIG. 11 shows the relative IL-23 and IFNα mRNA levels after stimulation of CD14.sup.+ monocytes with the indicated substances in the presence or absence of the TLR7/8 inhibitor ODN 2087.

[0061] FIG. 12 shows schematically the structure of a PNA-AntagomiR (3) coupled to CPP (1). The PNA-AntagomiR (3) is bound to the cell penetrating peptide (1) by means of a PEG linker (2).

[0062] FIG. 13 shows the results of an MTT assay for testing the cytotoxicity, wherein on the y-axis the viability is plotted. Neither in the case of the negative control (FIG. 13A) nor in the case of miR-574-5p PNA AntagomiR (FIG. 13B) a significant toxicity was observed.

[0063] FIG. 14 shows that the CPP-coupled miR-574-5p PNA AntagomiR results in a concentration-dependent reduction of the osteoclast genesis.

DETAILED DESCRIPTION OF THE INVENTION

Examples

[0064] Isolation of sEVs from the synovial fluid and the serum of ACPA+RA patients

[0065] From the synovial fluid of patients with rheumatoid arthritis who were positive for ACPAs (anti-citrullinated protein antibodies) sEVs (small extracellular vesicles, sEVs) were isolated. ACPAs are associated with a more severe course of the disease. The isolation of the vesicles was achieved with differential ultracentrifugation (FIG. 1).

[0066] With the help of transmission electron microscopy (TEM) it has been shown that the isolated population was characterized by the typical vesicular morphology and sEVs size of 30 nm to 150 nm. With Western blot, in addition, it has been determined that typical markers of sEVs (CD63, CD9, Hsp70 and CD81) were present. According to this, the isolated vesicles are sEVs.

[0067] In the same way, sEVs were also isolated from the serum of the patients.

[0068] Induction of osteoclast genesis by the vesicles isolated from the synovial fluid

[0069] CD14.sup.+ monocytes were isolated from PMBCs (peripheral blood mononuclear cells) of healthy donors and stimulated with M-CSF (recombinant human macrophage colony-stimulating factor), RANK-L (receptor activator of NF-κB ligand) and different concentrations of the isolated sEVs. With the sEVs isolated from the synovial fluid both freshly isolated monocytes and differentiated M2-like macrophages were stimulated (FIG. 2).

[0070] After 9 to 12 days the cells were fixed and tested for the osteoclast marker TRAP (tartrate-resistant acid phosphatase). TRAP positive cells which also comprised at least three nuclei were classified as osteoclasts and counted with the light microscope.

[0071] A significant concentration-dependent increase of the osteoclasts of about 30% was observed, when sEVs were added to monocytes (FIG. 3A). A similar increase was achieved, when the sEVs were added to M2-like macrophages (FIG. 3B). The results are indicated as mean value+SEM (standard error of the mean). The statistical analysis was conducted with an unpaired two-sided t test using the software GraphPad Prism 6.0. Differences were classified as significant for p<0.05 (indicated as * for p<0.05, ** for p<0.01, *** for p<0.001 and *′ for p<0.0001).

[0072] The results show that the isolated sEVs induce the osteoclast differentiation in a concentration-dependent manner.

[0073] High Content of miR-574-5p in Isolated Vesicles

[0074] With the help of RT-qPCR the content of different miRs in the isolated vesicles was determined. A high content of miR-574-5p (SEQ ID NO: 4) was determined in both the sEVs from the serum and the sEVs from the synovial fluid (FIGS. 4A and 4B). The results are indicated as mean value+SEM. On the other hand, the content of the other miRs tested (miR-16-5p, miR-155-5p and miR-146a-5p; SEQ ID NOs: 1-3) was considerably lower. The primers were purchased from the company Qiagen (Hilden, Germany) each (catalog number MS00043617 for miR-574; M50031493 for miR-16; MS00031486 for miR-155; and MS00003535 for miR-146a).

[0075] As a control for the normalization of the extracellular content non-human cel-miR39-3p (SEQ ID NO: 5) was used, which was added to the samples for this purpose in an end concentration of 200 nM. The primers also came from Qiagen (catalog number MS00019789).

[0076] The results show a selective accumulation of miR-574-5p in sEVs of patients with rheumatoid arthritis.

[0077] Synovial fibroblasts and monocytes as cellular sources of extracellular miR-574-5p

[0078] Synovial fibroblasts (SFs) were obtained from ACPA negative and ACPA positive patients with rheumatoid arthritis. Intracellular levels and sEV levels of the above-mentioned miRs were determined with RT-qPCR with and without stimulation for 24 hours with IL-1β(10 ng/ml) and/or TNFα (10 ng/ml) (FIGS. 5A and 5B). The sEVs were isolated from the supernatants of the cell cultures. For the normalization of the extracellular content always cel-miR-39-3p was used. For the normalization of the intracellular content always snRNA U6 (SEQ ID NO: 20) served as an endogenous control. The primers also came from Qiagen (catalog number M500033740).

[0079] miR-146a-5p served as a positive control, because its induction by stimulation with IL-1β is known (Stanczyk J., Pedrioli D. M. L., Brentano F., Sanchez-pernaute Q., Kolling C., Gay R. E., Detmar M., Gay S. and Kyburz D. (2008). “Altered Expression of MicroRNA in Synovial Fibroblasts and Synovial Tissue in Rheumatoid Arthritis.” Arthritis Rheum 58: 1001-1009). For the other miRs no significant stimulation-related differences could be observed.

[0080] Noticeable is the selective accumulation of miR-574-5p in the sEVs. While the intracellular content of miR-574-5p in the SFs in comparison to the intracellular content of miR-16-5p is considerably lower, in the isolated sEVs comparable contents of both miRs can be found (FIGS. 5A and 5B).

[0081] Interestingly, in addition, differences between SFs from ACPA negative (ACPA.sup.−) and ACPA positive (ACPA.sup.+) patients were observed. ACPAs are associated with a more severe course of the disease, and insofar they are suitable as an indicator of the severity of the course of the disease. As shown in FIG. 5D, in sEVs of SFs from ACPA positive patients in comparison to ACPA negative patients significantly increased amounts of miR574-5p were determined so that a connection can be established between the content of extracellular miR-574-5p and the severity of the course of the disease.

[0082] Besides SFs also M2 macrophages secrete miR-574-5p in sEVs (FIG. 6). While the intracellular level during the osteoclast differentiation substantially remains unchanged (FIG. 6A), a substantial secretion after 6, 9 or 12 days of differentiation does not take place any longer (FIG. 6B). Thus, pre-osteoclasts and osteoclasts do not substantially contribute to miR-574-5p in sEVs.

[0083] In summary, SFs and monocytes, however not pre-osteoclasts and osteoclasts contribute to miR-574-5p in sEVs.

[0084] sEVs with Designed miR-574-5p Level

[0085] With the help of XMIRXpress constructs (System Bioscience, Palo Alto, USA) in HEK 293 cells sEVs with different contents of miR-574-5p were designed. The content of miR-574-5p in the miR-574-5p oe sEVs was about 15 times higher in comparison to the control (ScrC sEVs) which was obtained with the help of a control miR (XMIRXP-NT, System Biosciences, Palo Alto, USA) (FIG. 7A).

[0086] In an RNase protection experiment it has been shown that the RNase degradation of the miR-574-5p can significantly be increased by an addition of a detergent (FIG. 7B). Thus, miR-574-5p is located in the vesicles which protect against RNase degradation, and it only becomes accessible for the degradation by RNase, when released by the detergent. This experiment shows that the miR-574-5p is mainly located in the sEVs and thus is protected against degradation by RNases. Only through the addition of the detergent a degradation of the miR is possible by the “destruction” of the sEVs.

[0087] Uptake of the Designed sEVs by Cells

[0088] miR-574-5p oe sEVs were stained with the fluorescent dye 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), and their uptake in CD14.sup.+ monocytes was examined by confocal microscopy. After 20 minutes an uptake of the stained vesicles in the cells was observed. The maximum number of stained sEVs in the cells was achieved after 40 minutes. Similar results were obtained for HeLa cells.

[0089] Induction of the Osteoclast Genesis by sEVs with High Content of miR-574-5p

[0090] Isolated human CD14.sup.+ monocytes were treated with 1 μg/ml miR-574-5p oe sEVs or ScrC sEVs at different time points of the differentiation (FIG. 8A, 8B, 8C).

[0091] The miR-574-5p oe sEVs result in a significant increase of the number of osteoclasts, when added to monocytes or M2-like macrophages (FIGS. 8A and 8B). Whereas in the case of an addition to pre-osteoclasts no significant increase of the number of osteoclasts was observed (FIG. 8C).

[0092] The ScrC sEVs also result in a significant increase of the number of osteoclasts, when added to monocytes or M2-like macrophages (FIGS. 8A and 8B). This is probably due to the miR-574-5p which is also present there, but the content of which in comparison to the miR-574-5p oe sEVs is lower by a factor of 15 (FIG. 7A) so that the stimulation of the osteoclast genesis with about 1.2-fold in the case of ScrC sEVs is considerably lower than in the case of miR-574-5p oe sEVs with about 1.7-fold.

[0093] The observed stimulation effect depends on the presence of miR-574-5p in sEVs. Neither miR-574-5p alone, nor miR-574-5p in the presence of synthetic liposomal vehicles (Lipofectamine® 2000 (Thermo Fisher Scientific, Waltham, USA)) results in a stimulation of the osteoclast genesis (FIG. 9).

[0094] Induction of the Osteoclast Genesis Mediated by Interaction of miR-574-5p with TLR7/8

[0095] In the context of the present invention, a direct interaction of miR-574-5p with TLR8 was detected by means of MST (microscale thermophoresis); (Wienken C. J., Baaske P., Rothbauer U., Braun D. and Duhr S. (2010). “Protein-binding assays in biological liquids using microscale thermophoresis.” Nat Commun 1: 100). The constant of dissociation K.sub.D was 30.8±5.2 nM (FIGS. 10A and 10B). However, a specific interaction between TLR8 and miR-16-5p was not shown (FIGS. 10A and 10B). For the MST experiments Cy5-labeled miR-574-5p (SEQ ID NO: 18) and Cy5-labeled miR-16-5p (SEQ ID NO: 19) were used.

[0096] An addition of the TLR7/8 inhibitor ODN 2087 (ODN 2088 Control (ODN2087), MiltenyiBiotec, Bergisch-Gladbach, GER) nullifies the above-described effect of miR-574-5p onto CD14.sup.+ monocytes and M2-like macrophages so that miR-574-5p in the presence of ODN 2087 can not result in a significant increase of the number of osteoclasts (FIGS. 10C and 10D).

[0097] Conversely, an increase of the osteoclast genesis can be achieved by an addition of the known TLR7/8 ligand R848 (Invivogen, San Diego, USA). But the effect of R848 strongly depends on the concentration. While in the case of 10 ng/ml a significant increase of the number of osteoclasts can be observed, a reduction of the number of osteoclasts arises in the case of 1000 ng/ml. 100 ng/ml of R848, when added to monocytes, result in an increase of the number of osteoclasts, whereas, when added to M2-like macrophages, in a decrease of the same (FIGS. 10E and 10F). It was possible to nullify the effect by an addition of the TLR7/8 inhibitor ODN 2087. As with miR-574-5p (FIG. 8C), no significant increase of the osteoclast genesis was observed, when the TLR7/8 agonist R848 was added to pre-osteoclasts.

[0098] The results shown in FIG. 10 verify that the effect of miR-574-5p oe sEVs onto the increase of the number of osteoclasts is mediated by an interaction with TLR7/8.

[0099] miR-574-5p Induces IFNα and IL-23 mRNA in CD14.sup.+ Monocytes by TLR7/8 Activation

[0100] CD14.sup.+ monocytes were stimulated with sEVs from the synovial fluid of ACPA positive patients with rheumatoid arthritis (4 μg/ml), with miR-574-5p oe sEVs (1 μg/ml) or with ScrC sEVs (1 μg/ml) for 4 hours each. Then the whole RNA was isolated and the mRNA levels of IL-23, IL-8, INFα, IL-1β and TNFα were analyzed with RT-qPCR. These cytokines are known for their influence onto the differentiation of osteoclasts (Amara-sekara D. S., Yun Ii., Kim S., Lee N. and Rho J. (2018). “Regulation of Osteoclast Differentiation by Cytokine Networks.” Immune Netw 18: [0101] 1-18). The sequences of the primer pairs used are shown in the sequence protocol (SEQ ID NOs: 6-7 for INFα, SEQ ID NOs: 8-9 for IL-23, SEQ ID NOs: 10-11 for TNFα, SEQ ID NOs:
12-13 for IL-1β and SEQ ID NOs: 14-15 for IL-8). For the normalization of the cDNA amounts in different samples the levels of β-actin were used (primer pair: SEQ ID NOs: 16-17).

[0102] The levels of TNFα, IL-1β and IL-8 were not influenced by miR-574-5p under any of the conditions tested. On the contrary, with a stimulation with sEVs which were obtained from the synovial fluid of patients with rheumatoid arthritis an about three-fold increase of the INFα mRNA could be observed (FIG. 118). Stimulation with miR-574-5p oe sEVs even resulted in an about five-fold increase of the INFα mRNA and in an about two-fold increase of the IL-23 mRNA, while with ScrC sEVs no increase was observed (FIGS. 11C and 11D). Comparable results were achieved by stimulation of the monocytes with 10 ng/ml of the TLR7/8 ligand R848 (FIGS. 11E and 11F). It was possible to nullify the observed effects by an addition of the TLR7/8 inhibitor ODN 2087.

[0103] Reduction of the Osteoclast Genesis by miR-574-5p AntagomiR Coupled to CPP

[0104] A miR-574-5p PNA AntagomiR was covalently bound via a PEG linker to a cell penetrating peptide (CPP) (see scheme in FIG. 12). The miR-574-5p PNA AntagomiR is a single stranded RNA analog which is complementary to miR-574-5p, namely a so-called “peptide nucleic acid” (PNA). The AntagomiR is completely complementary to miR574-5p, but it does not cover the whole sequence of miR-574-5p. The sequence of AntagomiR is shown in SEQ ID NO: 28. At the C-terminus the AntagomiR sequence was connected with a modified lysine. The modification consisted of the presence of an amide group (—CONH2) instead of the carboxyl group (—COOH). In the synthesis, the modified lysine is used for anchoring to the resin scaffold (see next paragraph). As a spacer with respect to the resin the modified lysine improves the efficiency of the first critical coupling. Furthermore, the modified lysine increases the solubility of the CPP-PNA construct.

[0105] Standard methods of solid phase synthesis were used. The whole CPP-PNA construct was built up on a resin scaffold, because all the synthesis building blocks used are compatible with each other. The synthesis was conducted from the C-terminus to the N-terminus. At first, the most C-terminal synthesis building block (here modified lysine) was immobilized on a resin scaffold. The next more N-terminal synthesis building block was then reacted with the synthesis building block which has been immobilized on the resin. This reaction cycle was repeated for each building block so that the macromolecule located on the resin grew building block by building block. When the most N-terminal building block has been reacted, then the finished CPP-PNA construct was released from the resin scaffold.

[0106] The CPP-PNA construct used can be illustrated as follows (from the N-terminus to the C-terminus): GRKKRWFRRRRMKWKK-(eg1)-ctcacacacacacactca(K—CONH2). The first part is the CPP (SEQ ID NO: 29). The expression (eg-1) describes the PEG linker which here comprises exactly one ethylene glycol unit. Then the PNA AntagomiR with SEQ ID NO: 28 follows. Terminatory, the construct contains a modified lysine with an amide group (—CONH2) instead of a carboxyl group (—COOH). The modified lysine is described by the expression (K—CONH2).

[0107] The cytotoxicity was tested with an MTT assay in which the eponymous dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) is used. As negative control a PNA with a sequence which was not complementary to miR-574-5p and which was also bound to CPP by means of the PEG linker was tested (SEQ ID NO: 30). The CPP-PNA construct of the negative control used can be illustrated as follows (from the N-terminus to the C-terminus): GRKKRWFRRRRMKWKK-(eg1)-gctattaccttaacccag(K—NH2). The negative control differs from the above-described construct according to the present invention only with respect to the PNA sequence.

[0108] The results are shown in FIG. 13, wherein on the y-axis the viability is plotted. Neither in the case of the negative control (FIG. 13A) nor in the case of miR-574-5p PNA AntagomiR (FIG. 13B) a significant toxicity was observed. In the case of miR-574-5p PNA AntagomiR even an increased viability in comparison to the untreated control was observed. The cytotoxicity was tested on freshly isolated human monocytes.

[0109] The influence of the miR-574-5p PNA AntagomiR which was coupled to CPP onto the osteoclast genesis was tested. The scheme of the induction of the osteoclast genesis is shown in FIG. 2. This scheme has been adapted such that on day 1 the CPP-coupled miR-574-5p PNA AntagomiR (CPP-PNA) was added in concentrations of 10 μM or 20 μM. The negative control was conducted without any addition of CPP-PNA (0 μm CPP-PNA). The results are shown in FIG. 14. The CPP-coupled miR-574-5p PNA AntagomiR results in a concentration-dependent reduction of the osteoclast genesis. Also for this experiment freshly isolated human monocytes were used. After the completion of the differentiation matured osteoclasts were visualized with the help of histologic staining (detection of tartrate-resistant acid phosphatases, TRAP). Subsequently, multinuclear TRAP positive cells are quantified under a light microscope.

LIST OF REFERENCE SIGNS

[0110] 1 cell penetrating peptide (CPP) [0111] 2 PEG linker [0112] 3 miR-574-5p PNA AntagomiR