The present invention relates to the use of N-Methyl-2-pyrrolidone for the prevention and treatment of bone loss.

Bone remodelling is a physiological process that involves the resorption and synthesis of bone by osteoclasts and osteoblasts, respectively. Osteoclasts are known to be formed by the fusion of hematopoietic cells of the monocyte-macrophage lineage during the early stage of the differentiation process. This process consists of multiple steps, including differentiation of osteoclast precursors into mononuclear osteoclasts, fusion of mononuclear preosteoclasts into mature multinucleated osteoclasts and activation of osteoclasts to resorb bone. The terminal differentiation in this lineage is characterized by acquisition of mature phenotypic markers such as expression of tartrate-resistant acid phosphatase (TRAP), calcitonin receptor, matrix metalloproteinase 9, and cathepsin K, as well as morphological conversion into large multinucleated cells and the ability to form resorption lacunae on bone. The essential signalling molecules for osteoclast differentiation include RANKL (receptor activator of nuclear factor κ-Β ligand) and M-CSF (macrophage colony-stimulating factor) in bone marrow-derived macrophage precursor cells. RANKL is a member of the tumour necrosis factor (TNF) superfamily that is expressed in osteoblasts. It interacts with the osteoclast cell surface receptor RANK, which in turn recruits TNFR-associated factors (TRAFs) and plays a crucial role in osteoclast differentiation.

Due to an aging society osteoporosis or other bone destructive disorders are a major problem in our health system. The major modalities currently used in osteoporosis treatment focus on decreasing bone destruction by reducing the formation and maturation of osteoclasts. Primarily they include estrogen replacement therapy along with the administration of bisphosphonates, selective estrogen receptor modulators (SERM), and calcitonin. Novel developments on this field are RANKL-binding antibodies (denosumab). However, such therapies are associated with adverse effects, including breast cancer, osteonecrosis of the jaw, hypercalcemia, and hypertension.

Therefore, substances or treatments able to reduce bone degradation and bone loss are needed.

The objective of the present invention is to provide safe and efficacious means for the prevention and treatment of bone loss, particularly in osteoporosis.

The surprising finding was made that N-methyl-2-pyrrolidone (NMP, CAS No. 872-50-4; formula I)

inhibits osteoclast differentiation by decreasing the expression of NFATd , and attenuates bone resorption by disrupting the actin rings and decreasing MMP 9 activity in multinucleated osteoclasts. The inhibitory effect of NMP on osteoclast maturation and function is independent from the previously known enhancing effect on bone regeneration, since the latter was described as enhancement of BMP activity in osteoblasts and preosteoblastic cells (Miguel et al, Tissue engineering 15 (2009) 2955-2963). In the context of bone degradation by osteoclasts it was shown by others that BMP stimulates osteoclast maturation and activity (Blackwell et al in Prostaglandins & other Lipid Mediators 90 (2009) 76-80 and Kaneko et al. in Bone 27 (2000) 479-486). Therefore an enhancement of BMP activity would foster osteoclast maturation and activity. In contrast, in this new patent application NMP was shown by the present inventors to do the opposite; to inhibit osteoclast maturation and activity. In terms of signaling pathways, the enhancement of bone regeneration occurred via BMP signaling and involved an increase in the phosphorylation of Smads and p38 and in short term increased phosphorylation of ERK as well. In osteoclasts, however, the present inventors detected no change in p38 phosphorylation but a decrease in ERK phosphorylation. Again, the effects of NMP on the signaling events in osteoclasts oppose the effects seen in osteoblasts and are therefore not related to it

Bone loss is caused by cancer, bone metastases, sepsis, rheumatoid arthritis, periodontitis, osteoporosis, osteoarthritis or other bone degrading disorders.

According to a first aspect of the invention N-methyl-2-pyrrolidone is provided for the prevention or treatment of bone loss.

Particular and preferred indications comprised in the above are N-methyl-2-pyrrolidone for the prevention or treatment of bone loss caused by cancer, particularly bone metastases, furthermore by sepsis, rheumatoid arthritis, periodontitis, osteoporosis or other bone degradation disorders.

According to another aspect of the invention, a dosage form is provided for the prevention or treatment of bone loss, particularly osteoporosis, comprising N-methyl-2-pyrrolidone. Optionally, a pharmaceutical carrier or excipient may be present. N-methyl-2-pyrrolidone can be applied to a subject in any form suitable to the intended treatment. Such a form may be an oral formulation, nasal inhalant, injection, a suppository or a topical formulation. A topical formulation, particularly an ointment or dermal patch, or an injectable formulation is preferred. According to a preferred embodiment of all aspects of the invention, N-methyl-2-pyrrolidone may be applied locally. In this case a pharmaceutically acceptable carrier is a slow release system based on synthetic biodegradable polymers, such as polyglycolide, polylactides, polycaprolactones, polytrimethylenecarbonates, polyhydroxybutyrates, polyhydroxyvalerates, polydioxanones, polyorthoesters, polycarbonates, polytyrosinecarbonates, poly- orthocarbonates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(maleic anhydride), polypeptides, polydepsipeptides, polyvinylalcohol, polyesteramides, polyamides, polyanhydrides, polyurethanes, polyphosphazenes, polycyanoacrylates, polyfumarates, poly(amino acids), modified polysaccharides (like cellulose, starch, dextran, chitin, chitosan, etc.), modified proteins (like collagen, casein, fibrin, etc.) and their copolymers, terpolymers or combinations or mixtures or polymer blends thereof. Polyglycolide, poly(L-lactide-co-glycolide), poly(D,L-lactide-co-glycolide), poly(L-lactide), poly(D,L-lactide), poly(L-lactide-co-D,L-lactide), polycaprolactone, poly(L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone) polytrimethylenecarbonate, poly(L-lactide-co-trimethylenecarbonate), poly(D,L-lactide-co- trimethylenecarbonate), polydioxanone and copolymers, terpolymers and polymer blends thereof are highly preferred examples of polymers. Most preferably, the copolymer of polylactide with glycolides, such as poly-DL-lactide-co-glycolide (PLGA), is used as a carrier.

Other examples of pharmaceutically acceptable carriers useful in the present invention include proteins, like collagen and fibrin, calcium phosphates (tri-calciumphospates, hydroxyapatite), bone cements, bioglass, coral minerals (Algipore®), nacre, egg shell, bovine derived hydroxyapatite (Bio-Oss®), and all osteoconductive (porous) materials.

In one embodiment of the invention the pharmaceutical composition is the coating of an implant, or an implant itself, the implant including membranes, films, plates, mesh plates, screws tabs and other formed bodies.

Similarly, N-methyl-2-pyrrolidone may be comprised in a bone cement or filler used, for example, to substitute bone mass after removal of tumour tissue in bone, particularly in a vertebra.

According to yet another aspect of the invention, a treatment regime is provided for the prevention and treatment of bone loss, comprising the administration of N-methyl-2- pyrrolidone. Administration may be effected by any of the means described herein.

Also within the scope of the present invention is a method for the prevention or treatment of bone loss, comprising the administration of N-methyl-2-pyrrolidone to a subject in need thereof.

Similarly, N-methyl-2-pyrrolidone is provided for the manufacture of a medicament for the prevention and treatment of bone loss. Medicaments according to the invention are manufactured by methods known in the art, especially by conventional mixing, coating, granulating, dissolving or lyophilizing. The invention is further illustrated by the following figures and examples:

Brief description of the figures:

Figure 1 shows the inhibition of osteoclastogenesis in dependence of NMP dosage.

Figure 2 shows the absence of cytotoxic effects of NMP on the osteoclastogenesis of Raw 264.79 cells.

Figure 3 shows the suppression of the osteoclast differentiation by NMP and decrease of the fusion process.

Figure 4 shows the inhibition of RANKL-induced bone resorption by NMP.

Figure 5 shows the disruption of RANKL-induced actin ring formation by NMP.

Figure 6 shows the inhibition of RANKL-induced MMP 9 and cathepsin K expression by NMP. Figure 7 shows the suppression of the RANKL-induced NFATd expression by NMP.

Figure 8 shows the inhibition of the RANKL-mediated c-Fos protein expression by NMP.

Figure 9 shows a schematic model of action of NMP in osteoclastogenesis.

Detailed description of the invention

The present invention is based on the results of a clinical trial on 10 patients where a NMP releasing guided bone regeneration membrane was applied on the test side and a membrane without NMP on the control side after the removal of wisdom teeth on both sides. Based on the known osteopromotive effect we had expected that in the histologies new bone density would be increased in the samples exposed to NMP, since NMP is an enhancer of BMP activity. But an enhancement of BMP activity could also trigger the maturation and activity of osteoclasts and with it decrease bone mineral density. Unexpectedly, the histological results showed an increase in old bone density in the extraction socket model from 3.78±2.68 arbitrary units without NMP to 9.48±2.97 arbitrary units in the sockets treated with the NMP releasing membrane, indicating that NMP inhibited the degradation of the old bone and pointing to an inhibiting effect on osteoclast activity. An inhibitory effect on osteoclast maturation and activity has not been reported before and can not be derived from a stimulation of BMP activity and signaling, since this would enhance osteoclast maturation and activity and would not be in line with the present unexpected findings. To substantiate these findings primary osteoclasts or a model cell line for osteoclasts (RAW264.7 cells) was used. For both cell types we found that, indeed, NMP is able to inhibit osteoclastogenesis, the differentiation to osteoclasts, which is needed for bone degradation. RAW264.7 cells were chosen because they are a good model system to study osteoclastogenesis. Treatment of RANKL-stimulated RAW264.2 cells with increasing concentration of NMP inhibited the formation of multinucleated osteoclasts (Fig.1 ). In the same concentration range, NMP had no cytotoxic effect on the osteoclastic cells (Fig. 2).

The tartrate-resistant acid phosphatase (TRAP) is needed to generate the acidic milieu to dissolve the calcium-phosphates of bone. The examination of the effect of NMP on TRAP activity induced by RANKL (Fig. 3) revealed that TRAP activity was significantly reduced in the cells treated with RANKL and NMP, compared to the cells treated with RANKL alone (Fig. 3A). Moreover, NMP dramatically reduced the number of nuclei per osteoclast suggesting that NMP could modulate also the fusion process (Fig. 3B).

The effect of NMP on the ability of mature osteoclasts to resorb bone was evaluated in RAW 264.7 cells plated on bone slices and stimulated with RANKL in the presence or absence of NMP. RANKL-stimulated cells formed a number of pits, suggesting that the bone resorption activity of RANKL-treated cells transformed them into functionally active state resembling osteoclasts (Fig. 4). Treatment with 5 mM NMP significantly reduced the formation of resorption pits in numbers and in overall area as compared to treatment with RANKL alone. Bone resorption occurs within the sealing zone, which is formed by an actin ring structure. In order to investigate the effect of NMP on actin ring formation, immunofluorescence analysis was performed (Fig. 5). The majority of RANKL treated cells revealed well-formed actin rings (Fig. 5A-a). Cells treated with RANKL in the presence of 5 mM NMP displayed mainly disrupted actin rings (Figure 5A-b). As expected, cells treated with RANKL in the presence of 10 mM NMP showed no actin rings compared to cells treated with RANKL alone. It has been reported that osteoclasts displaying a full actin ring or disrupted actin rings with more than 50% intact were identified as active. The results are in line with this observation since with 1 mM NMP concentration, which is ineffectively inhibited osteoclast differentiation, the actin ring is not completely disrupted (Figure 5B).

MMP-9 and cathepsin K are bone resorption-related enzymes mainly involved in the degradation of the bone forming proteins. They are highly expressed in osteoclastic cells and play an important role in skeletal remodelling. RAW264.7 cells treated with RANKL showed a concentration-dependant increase in MMP 9 gelatinolytic activity (Fig. 6A). This gelatinolytic activity was significantly decreased by NMP treatment and was correlated with the suppression of osteoclast differentiation visualized when the same culture were stained for TRAP (data not shown). Figure 6B demonstrates that RANKL induced an increase in MMP 9 mRNA and only treatment with 10 mM NMP was able to decrease the RANKL-induced MMP 9 mRNA. Cathepsin K mRNA expression was increased by RANKL treatment. In the presence of NMP (5 and 10 mM), RANKL-induced cathepsin K mRNA was significantly suppressed (Fig. 6C).

NFATd , a member of the NFAT family of transcription factor, has been shown to be up- regulated after RANKL stimulation and to be important for osteoclast differentiation. Therefore, the effect of NMP on the expression of NFATd was examined (Fig. 7). RAW264 cells were stimulated with RANKL alone or with RANKL and different concentrations of NMP as indicated on the figure (48h for mRNA and 72h for protein). The stimulation of RAW264.7 cells with RANKL induced a high level expression of NFATd mRNA (Figure 7A). The treatment with NMP (5 mM) significantly decreased RANKL-induced NFATd mRNA. RANKL-induced NFATd mRNA was completely blocked by 10 mM of NMP. As shown in Fig. 7B, cellular protein expression was correlated with mRNA expression. In this case, the RANKL-induced NFATd expression was significantly and concentration-dependently attenuated by NMP treatment. With 10 mM NMP, the expression of NFATd induced by RANKL becomes similar to untreated cells.

c-Fos, a transcription factor for AP-1 complex, is required for osteoclast differentiation. C-Fos has been shown to bind to the promoter site of NFATd . c-Fos is important for osteoclast differentiation and NFATd activation. RAW264.7 cells were treated with RANKL or RANKL and NMP as described on the figure. Protein level was analyzed by western-blot using c-Fos and total ERK antibodies. As shown in figure 8, c-Fos expression was increased by RANKL, and NMP treatment (5 mM) significantly decreases c-Fos expression induced by RANKL.

In summary NMP suppresses c-Fos and NFATd expression, which are important downstream regulators of osteoclast differentiation. NMP disrupts actin ring formation possibly by inhibiting c-Src activation. Thus, NMP inhibits osteoclast differentiation by modulating various signalling molecules independent of the BMP signalling pathway.

The invention is illustrated by the following examples.

For statistical analysis, unpaired Students T-test was implemented by a commercially available software package (SSPE, Chicago, II). All values are represented as mean ± standard error of the mean.

Example 1 : NMP suppresses RANKL-induced osteoclastogenesis in RAW264.7 cells. To show the effects of NMP on osteoclastogenesis, RAW 264.7 cells were treated with RANKL (25 and 50 ng/ml) (Fig. 1). RAW 264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin). The cultures were never allowed to become confluent. Incubations were performed at 37°C in 5% C0 ₂ in humidified air. RAW264.7 cells were seeded on a 12-well culture plate, treated for 6 days with NMP, RANKL or NMP+RANKL as indicated on figure (1). Cells were washed with PBS, fixed, and stained for TRAP. Stained cells were photographed, and TRAP(+) MNCs containing three or more nuclei were counted as osteoclasts. Data are expressed as mean S.D. (n=3). In the absence of NMP, RAW264.7 cells differentiate into mature TRAP-positive multinucleated osteoclasts (MNCs), while NMP (5 and 10 mM) reduced the formation and numbers of TRAP-positive MNCs in a concentration-dependent manner (Fig. 1A). The number of MNCs was quantified microscopically (Fig. 1 B). In the presence of NMP (5 mM), the MNCs number induced by RANKL (25 ng/ml) was reduced approximately by 50%, whereas 10 mM NMP abolished totally the formation of MNCs.

To exclude the possibility that the inhibition was due to cytotoxicity of NMP, cell cytotoxicity/viability was analyzed using a non-radioactive WST-1 Assay (Fig. 2). Cells were seeded on a 96-well plate, treated for 24h or 72h with RANKL alone (25 ng/mL), or RANKL with different concentration of NMP. The effect of different concentrations of NMP on RAW 264.7 cells proliferation / viability was analyzed using a non-radioactive WST-1 Cell Proliferation Assay kit (Roche Diagnostics) according to the manufacturer's instructions. The results were normalized to cells grown in DMEM alone (control). Columns represent the mean of four replicates per condition from a representative experiment that was repeated three times with similar results; bars, SD. NMP demonstrated no cytotoxic effects after 24 and 72 h treatment with the concentration which effectively inhibited osteoclast formation.

Furthermore the effect of NMP on TRAP activity induced by RANKL was examined (Fig. 3). RAW264.7 cells were seeded on a 24-well culture plate, treated with RANKL alone or with different concentration of NMP as indicated on the figure. After 6 days incubation, cells were washed with PBS, fixed, and stained for TRAP after differentiation into osteoclasts. In parallel, TRAP activity was measured. Data are expressed as mean S.D. (n=3). For determination of TRAP activity and for TRAP-staining RAW 264.7 cells were plated into a 12-well culture dish (Corning, NY) with different concentrations of NMP in the presence of 25 ng/ml of RANKL. The medium and factors were replaced every 2 days. After 6 days of culture, the medium was removed and the cell monolayer was gently washed twice with PBS. The cells were then lysed with 200 μΙ of 0.1 % Triton X-100. TRAP activity in cell lysate was determined using TRAP solution (sodium acetate 0.1 M pH 5.8, ascorbic acid 1 mM, KCI 0.15 M, disodium tartrate 10 mM and p-nitrophenyl phosphate 10 mM). An aliquot of cell lysate was added to TRAP solution and was incubated for 30 minutes at 37°C. The reaction was stopped with NaOH 0.3 N and absorbance was measured at 410 nm using a micro-plate reader (BioTek). Results, normalized to protein content, are expressed in percentage of the activity obtained in RANKL stimulated cells. TRAP histochemical staining of the cells was performed using a leukocyte acid phosphatase kit (Sigma-Aldrich). Cultured cells were fixed with formaldehyde for 5 min at room temperature, washed with PBS and air-dried. After TRAP-staining, TRAP-positive multinucleated (more than 3 nuclei) cells were counted under phase-contrast microscopy. The TRAP activity was significantly reduced in the cells treated with RANKL and NMP compared to the cells treated with RANKL alone (Fig. 3A). Moreover, NMP dramatically reduced the number of nuclei per osteoclast suggesting that NMP could modulate the fusion process (Fig. 3B, photomicrographs of TRAP (+) cells). The results of this example show, that exposure of osteoclast precursor cells to NMP inhibit their RANKL induced maturation to osteoclasts.

Example 2: NMP attenuates bone resorption by disrupting actin ring formation and decreasing MMP-9 activity.

NMP inhibits bone resorption and actin ring formation. It was further examined if NMP has an effect on the ability of mature osteoclasts to resorb bone (Fig. 4). RAW 264.7 cells were plated on bone slices and stimulated with RANKL in the presence or absence of NMP. In detail RAW 264.7 cells were placed on bone slices in 24-well plates. After preincubation for 6 h, the bone slices were transferred to 12-well plates (1 bone slice/well) and further cultured in the presence or absence of 25 ng/ml RANKL and 5mM NMP. The medium was replaced with a fresh one every 2 days. After 9 days of culture, cells were completely removed and the bone slices were stained with hematoxylin or toluidine blue and quantified by image analysis system followed by photography. Histograms represent the percentage of resorbed area. Resorption pits of representative cultures are shown. RANKL-stimulated cells formed a number of pits, suggesting that the bone resorption activity of RANKL-treated cells made them into functionally active state resembling osteoclasts (Fig. 4). Treatment with 5 mM NMP significantly reduced the formation of resorption pits in numbers and in overall area as compared to treatment with RANKL alone.

Bone resorption occurs within the sealing zone, which is formed by an actin ring structure. In order to investigate the effect of NMP on actin ring formation, immunofluorescence analysis was performed (Fig. 5). For this purpose RAW 264.7 cells were differentiated in the presence of RANKL alone or with RANKL and 1 , 5 or 10 mM of NMP for 6 days. After this period cells were fixed. Actin rings of osteoclasts were detected by staining actin filaments with rhodamine-conjugated phalloidin. Osteoclasts were formed from RAW 264.7 cell cultures in the presence of RANKL (25 ng/mL) and NMP (1 , 5 and 10 mM). At the end of incubation, osteoclasts were stained with rhodamin-conjugated phalloidin and DAPI for nucleus. The distribution of actin rings was visualized and detected under a fluorescence microscope. The majority of RANKL treated cells revealed well-formed actin rings (Fig. 5A-a). Cells treated with RANKL in the presence of 5 mM NMP displayed mainly disrupted actin rings (Fig. 5A-b). Cells treated with RANKL in the presence of 10 mM NMP shows no actin rings compared to cells treated with RANKL alone.

NMP suppressed RANKL-induced MMP 9 and cathepsin K. Since MMP-9 and cathepsin K, bone resorption-related enzymes, are highly expressed in osteoclastic cells and plays an important role in skeletal remodelling, the effect of NMP on the expression of MMP 9 and cathepsin K mRNA was investigated. Additionally the MMP 9 activity was evaluated by gelatine zymography. RAW264.7 cells treated with RANKL showed a concentration- dependant increase in MMP 9 gelatinolytic activity (Fig. 6A). Both proteins are needed for the degradation of the proteins of bone. mRNAs were determined by real-time PCR after 48h stimulation with RANKL or RANKL in the presence of NMP (as indicated in the figure). RNA from Raw26437 cells was extracted using the RNeasy kit from Qiagen. The mRNA was reverse transcribed into cDNA cDNA and the resultant cDNA was subjected to real-time PCR with gene-specific primers using an iCycler real-time PCR machine using iQ SYBR Green super-mix (both from Bio-Rad Laboratories) according to the manufacturer's instructions. For protein preparation and Western blot analysis RAW264.7 cells treated with the different compounds were rapidly frozen in liquid nitrogen and stored at -80°C for further analysis. Cells were lysed as described in N. Ishida et al.,, J Biol Chem 277 41 147-41 156, 2002. Proteins were fractionated onto a 12% SDS polyacrylamide gel electrophoresis, transferred to Immobilon P membranes (Millipore), and immunoblotted with specific antibodies. Detection was performed using peroxidase-coupled secondary antibody, enhanced chemiluminescence reaction (Amersham ECL Western Blotting Detection Reagents, GE Healthcare Europe GmbH, Otelfingen, Switzerland), and visualization using autoradiography. Membranes that were reprobed had been stripped in stripping buffer (62.5 mM Tris-hydrochloric acid, pH 6.8, 2% (w/v) SDS, 100 mM β-mercaptoethanol) according to the manufacturer's protocol (Millipore). For zymography equal volumes of protein were loaded onto a 10% SDS-PAGE gel containing 0.1 % porcine gelatin (Sigma). After electrophoresis, the gels were washed twice for 15 min each in 2.5% Triton X-100 and then incubated overnight at 37 °C in substrate buffer (50 mM Tris-HCI, 0.2 M NaCI, 5 mM CaCI ₂ and 0.02% Brij 35). The gels were stained with Coomassie Blue R-250 (Sigma) for 1 h. The gels were de-stained briefly in 50% methanol, 10% acetic acid. Areas of gelatinolytic activity appeared cleared against the blue background of the blue- stained undigested gelatin.

This gelatinolytic activity was significantly decreased by NMP treatment and was correlated with the suppression of osteoclast differentiation visualized when the same culture were stained for TRAP (data not shown). Figure 6B demonstrate that RANKL induced an increase in MMP 9 mRNA and only treatment with 10 mM NMP was able to decrease the RANKL- induced MMP 9 mRNA. Cathepsin K mRNA expression was increased by RANKL treatment. In the presence of NMP (5 and 10 mM), RANKL-induced cathepsin K mRNA was significantly suppressed (Fig. 6C).

In essence, NMP disrupts actin ring formation and decreases MMP-9 activity in osteoclasts. Therefore NMP stops bone degradation.