Field of Invention:

The Invention relates to the clinical management of Osteoarthritis. Particularly, the Invention provides Disease modifying agents, Drug delivery system and method thereof for the management of Osteoarthritis.

Background of the invention:

The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Osteoarthritis is the most common musculoskeletal disorder in India that affects cartilage that lines the joints and is a leading cause of disability and locomotive discomfort. Articular cartilage is a specialized tissue that is a vascular, alymphatic, hypocellular and provides a smooth, lubricated surface for frictionless locomotion at the bone ends. During osteoarthritis articular cartilage becomes bony, vascularized and undergoes progressive irreversible deterioration. The development of articular cartilage and the maintenance during adult life is poorly understood. During embryonic limb skeleton development, the long bones develop from a common cartilage template the majority of which is mineralized and known as transient cartilage while that the extremities remains as cartilage during postnatal life. Moreover, the cell population that gives rise to these two distinct tissues comes from a common pool. The molecular basis of simultaneous differentiation of articular and transient cartilage was poorly understood till Ray et al. demonstrated that it is a spatio-temporal interplay between BMP and Wnt signaling that facilitates the differentiation of articular cartilage. Wnt signaling promotes differentiation of articular cartilage while BMP signaling promotes transient cartilage differentiation. The joint site is a source of Wnt ligands while no active BMP signaling operates in this region and there is a domain of Noggin expression, which restricts the BMP signaling to transient cartilage region. Moreover, the expression of BMP signaling and Wnt signaling in articular cartilage is highly regulated by mechanical loading and a disruption of the mechanics at the joint site leads to ectopic transient cartilage differentiation in place of articular cartilage differentiation which is mediated by an upregulation of BMP signaling.

At present, there is no disease modifying agent available to treat osteoarthritis in any drug market. This is an alarming scenario considering the fact that most epidemiological studies predict a steep rise in the prevalence of osteoarthritis in the near future. Moreover, current treatments focus only on symptomatic management for inflammation and pain, which does halt the progression or severity of this disease. This is the biggest pain point that can be resolved using this innovation. Hence, there is need of an effective Disease modifying agents, Drug delivery system and method thereof for the management of Osteoarthritis.

Objectives of the Invention:

Primary object of the present invention is to overcome the limitation associated with the prior art.

Yet another object of the present invention is to provide an effective solution for the clinical management of Osteoarthritis.

Yet another object of the present invention is to provide Disease modifying agents, Drug delivery system and method thereof for the management of Osteoarthritis.

Yet another object of the present invention is to provide molecules halting or reversing the osteoarthritis.

Yet another object of the present invention is to provide BMP inhibitors for the management of osteoarthritis.

Yet another object of the present invention is to provide bio materials in which BMP inhibitor can be encapsulated and delivered in the joints where a sustained release of BMP inhibitor can be achieved.

Yet another object of the present invention is to provide injectable bio materials for the controlled release of BMP inhibitor in the joints. Detailed Description of Drawings:

To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings in which:

Figure 1: illustrates the experiment conducted for investigating sufficiency of BMP signaling in inducing osteoarthritis

Figure 2: illustrates activation of BMP signalling in adult mouse articular cartridge

Figure 3: illustrates activation of BMP signalling in adult mice articular cartridge

Figure 4: illustrates the experiment conducted for investigating if transient cartilage differentiation induced by BMP signaling is necessary for the development of osteoarthritis

Figure 5: illustrates schematic for ACLT experiments

Figure 6: illustrates effect of deletion of BMP signalling in a mice surgical model of osteoarthritis

Figure 7: illustrates effect of local administration of BMP inhibitor to halt osteoarthritis progression

Figure 8: illustrates osteoarthritic changes resulting from ACLT can be counteracted by inhibiting BMP signalling by LDN.

Figure 9: illustrates cumulative release profile of LDN- 193189 from injectable hydrogels (1%), a) 10 pg of LDN- 193189 loaded per 100 pL of hydrogel, b) 50 pg of LDN-193189 loaded per 100 pL of hydrogel.

Figure 10: illustrates stability and activity of released LDN-193189 from injectable hydrogels upto 6-days by inhibiting the luciferase expression in BRITER cell lines. Figure 11: illustrates (a) LDN-193189 loaded k-carrageenan hydrogel injection in joint cavity attenuates osteoarthritic changes in mice articular cartilage (b) illustrates LDN-193189 loaded k- carrageenan hydrogel injection in joint cavity perpetuate articular cartilage integrity for longer duration in osteoarthritic mice model

Detailed description of the invention:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof. Throughout the patent specification, a convention employed is that in the appended drawings, like numerals denote like components

The Invention relates to the clinical management of Osteoarthritis. Particularly, the Invention provides Disease modifying agents, Drug delivery system and method thereof for the management of Osteoarthritis.

The Invention, in one of its aspect, has performed a comprehensive analysis of molecular markers in articular cartilage during osteoarthritis which reveals that articular cartilage starts expressing molecules and matrix components similar to those found in articular cartilage. Moreover, the progression of osteoarthritis follows molecular events similar to transient cartilage differentiation which suggests that osteoarthritis is essentially a trans-differentiation of articular cartilage into transient cartilage during post-natal stages. Since BMP signaling is essential for transient cartilage differentiation, it is highly likely that BMP signaling could promote this process during the pathogenesis of osteoarthritis as a disruption of mechanical loading triggers the initiation of degradation of articular cartilage. In an embodiment, the invention provides molecules halting or reversing the osteoarthritis.

In an embodiment, the invention provides BMP inhibitors for the management of osteoarthritis.

In an embodiment, the invention provides injectable biomaterials for the controlled release of BMP inhibitor in the joints.

In an embodiment, the invention provides biomaterials in which BMP inhibitor can be encapsulated and delivered in the joints where a sustained release of BMP inhibitor can be achieved.

In an embodiment, the invention provides an ionic pair of inhibitor and delivery vehicle which allows to have a prolonged sustained release. The delivery system proposed facilitate scaffold for cartilage tissue growth due to its mechanical properties which microparticle formulation lacks. The mechanical properties and inhibitor loading in injectable delivery systems can be tuned as per demand.

In an embodiment, the invention provides a method of intra-articular administration which has an advantage of delivering inhibitor on site with required therapeutic index and reduces off target systemic drug distribution,

In an embodiment the Invention provides BMP inhibitor for the treatment of osteoarthritis comprising LDN-193189 wherein LDN-193189 is present in the range of lOuM to 50 uM.

In an embodiment, the Invention provides a carrier vehicle for intra-articular injection of the BMP inhibitor as claimed in claim 1, comprising 3% 2-(hydroxypropyl)- b- cyclodextrin (w/v) in sterile 1% w/v PBS.

In an embodiment the Invention provides an injectable hydrogel composition for the treatment of osteoarthritis comprising LDN-193189 loaded k-carrageenan hydrogel in 1% w/v PBS wherein k-carrageenan is present in an amount ranging from 0.4 to 20% w/v and wherein approx 1 pg to 100 pg of LDN-193189 is present per 100 pL of hydrogel composition.

In an embodiment, LDN-193189 loaded k-carrageenan hydrogel is present in an amount ranging from 6pl -8pl. In an embodiment the Invention provides a method of preparing injectable hydrogel composition, as described above, for the treatment of osteoarthritis comprising: a) 0.4 to 20% w/v of polymer solution was prepared in IX PBS by heating at 70°C until dissolution at temperature ranging from 45°C to 90°C; where said polymer solution comprises k-carrageenan, alginic acid, chitosan, hyaluronic acid, collagen, gelatin, preferably k-carrageenan; b) adding 35 pL of LDN-193189 in approx 2 mg/mL of DMSO to k- carrageenan solution obtained from step (a) followed by vortexing to make a homogenous solution; c) aspirating the solution obtained from step (b) in 30G syringe and leaving for overnight for gelation wherein said resulting hydrogel comprises approx 1 pg to 100 pg of LDN-193189 per 100 pL of hydrogel.

In an embodiment, the polymer solution is prepared by dissolving 7 mg of k-carrageenan solution in 665 pL of IX PBS.

In an embodiment the Invention provides a method of administering BMP inhibitors for the treatment of osteoarthritis, wherein said administration comprises intra-articular injection of LDN-193189 loaded k-carrageenan hydrogel (1% w/v in PBS), as described above, having concentration of 500ng/pl wherein the k-carrageenan is present in an amount ranging from 0.4 to 20% w/v.

In an embodiment, the method of administration comprises administering LDN-193189 where first dose was administered at the first day and seven consecutive doses were administered starting at day 14 post surgery and continued till day 21 post surgery.

In an embodiment, the method of administration comprises administering LDN-193189 encapsulated k-carrageenan hydrogel administered once after the surgery.

The major advantage of present hydrogel technology is reducing the frequency of injections. Typically, intraarticular injections are permitted in human only twice in a year to prevent injection-induced knee damage. Therefore, daily injection of free-drug is clinically impossible. Therefore, the hydrogel of the present invention has a great advantage in clinical application. Our mice data suggests that even single injection has efficiency to reduce the disease. Additionally, two doses (once in month) gave similar efficacy.

In an embodiment, the method comprises single administration of LDN-193189 encapsulated k-carrageenan hydrogel.

In another embodiment, the method comprises administration of LDN-193189 encapsulated k-carrageenan hydrogel once in three or six months.

In an embodiment, the Invention provides a method of treating osteoarthritis comprising administering LDN-193189 in an amount ranging from lOuM to 50 uM.

The Invention is further described with the help of non-limiting examples:

Example 1:

To test whether BMP signaling is sufficient for pathogenesis of osteoarthritis, the experiment was conducted to overexpress the constitutive ly active BMP receptor type 1A (caBMPRIA) in articular cartilage specifically during postnatal stages in mice. The experiment used the Cre- LoxP system to induce recombination specifically in articular cartilage using a Collagen type 2 promoter (Col2al- Cre) and the temporal specificity was mediated by Tamoxifen injection as the Cre recombinase is fused to a modified Estrogen receptor. The experiment crossed a mice strain possessing a constitutively active BMP receptor la (pMes- caBMPRIA) with the Col2al- Cre- ERT2 strain and induced recombination at P56 by intra-peritoneal administration of tamoxifen and it was observed that ectopic activation of BMP signaling in articular cartilage leads to an osteoarthritis like phenotype.

The Anterior cruciate Ligament transection is a well-established surgical model in mice and higher mammals to induce mechanical instability which leads to the development of osteoarthritis post-surgery. In this technique, one of the four key ligaments which support the knee joint and maintain the mechanics is transected which leads to disruption of the loading on the joint. To transect the Anterior cruciate ligament, the patellar tendon is bypassed and synovial cavity is opened to expose the ligament and the transection is done by pulling the ligament with a needle and cutting with a spring loaded scissor. In the sham surgery, the incision is made to open the knee space which is then sutured without transecting the ligament. ACLT is performed at PI 20 stage in mice and then knees are harvested at different time points post surgery.

The median time when overt signs of phenotype appear is ~3 weeks post-surgery. It was found that the osteoarthritis resulting from surgical induction also resulted in transient cartilage differentiation. Moreover, it was also observed that there is an upregulation of pSmadl/5/8 i.e. active BMP signaling in articular cartilage following upon ACL transection while no active BMP signaling is observed in the articular cartilage of the sham operated animal. The Inventors conducted a time course study to study the kinetics of transient cartilage differentiation and to ascertain the earliest time where and if at all BMP signaling is upregulated. In this experiment, it wad found that pSmad 1/5/8 expression spikes in articular cartilage at day 7 post surgery and is overtly evident at day 14 post ACLT. This precedes the major transient cartilage differentiation events viz. the expression of Indian hedgehog which marks pre -hypertrophic cells, Collagen type X which is a marker of hypertrophy, matrix remodeling by enzymes such as MMP-13, ADAMTS-5 and vascular invasion mediated by VEGF. Moreover, aggrecan degradation is another hallmark of osteoarthritis induction which is ascertained by the presence of a neo -epitope NITEGE generated when ADAMTS-5 cleaves aggrecan core at certain amino acid residues.

Example 2:

In order to test the necessity of BMP signaling in the development of osteoarthritis, the Inventors used a surgical model to induce osteoarthritis in mice wherein the anterior cruciate ligament (ACL) in the knee was transected which induces mechanical instability that ultimately leads to development of osteoarthritis in mice. To this end, the present invention uses a conditional mouse strain which can be depleted of BMP ligands BMP2 and BMP4 (Bmp2/4 DCKO) with spatial and temporal specificity in articular cartilage (Bmp2/4 ^c/c ; Col2al-Cre-ERT2) during postnatal stages upon tamoxifen administration. The Inventors performed ablation of BMP signaling by administering three doses of tamoxifen (100 mg/kg) at P56 and ACLT was performed at P120. The tissues were harvested at time points 28 days and 56 days post surgery. Upon performing ACLT in mice which had been depleted of BMP ligands Bmp2 and Bmp4, it was found that the severity of osteoarthritis was significantly attenuated as compared to the ACLT alone mice.

Example 3:

These results suggested that the local inhibition of BMP signaling could be an approach to halt the degradation of articular cartilage. LDN-193189, an analogue of dorsomorphin, which possesses high selectivity and potency to inhibit canonical BMP signaling mediated by pSmad 1/5/8. LDN -193189 is obtained by modifying the pyrimidine ring and replacing it with 4- quinolinyl group which led to higher potency as compared to dorsomorphin. It has been shown to reverse the heterotopic ossification observed in Fibrodysplasia ossificans progressiva phenotype where constitutive activation of BMP signaling leads to ossification of soft tissues upon injury or trauma by Mishina et al. This makes it an ideal candidate for use in this context where trans- differentiation of articular cartilage into transient cartilage or bone occurs. The IC50 value of LDN-193189 is 4nM for inhibition of BMP receptor 1A and 50nM for the BMP receptor IB. The concentration used by Mishina et al. for intraperitoneal administration was 3 mg/kg body weight. LDN-193189 was locally administered at the above mentioned dose and seven consecutive doses were administered starting at day 14 post surgery and continued till day 21 post surgery. The Tissue samples were collected at day 28 post surgery.

Example 4:

The route of administration of LDN-193189 used in this study was intra- articular injection into the knee joint space. LDN-193189 injection solution was delivered in the intra-articular space using 3% 2-(hydroxypropyl)- b- cyclodextrin (w/v) in sterile PBS as the carrier vehicle. The intra-articular space for mice knee joint was injected by LDN-193189 where LDN-193189 is present in amount of ~6ul - 8 ul and the concentration of LDN-193189 used was lOuM. This concentration was decided considering the minimal amount of knee space available and the IC50 of the compound. The animal was anesthetized using isoflurane and 2% O2 (medical grade) and the knee joint was flexed to expose the knee cavity which was ascertained by nudging to find the depression of the synovial cavity. The payload was injected using a 30G syringe till the synovial cavity was flush with the solution and a single injection was performed for the duration of the regimen. It was observed that local administration of LDN-193189 attenuated the osteoarthritic phenotype post ACLT.

Example 5: Hydrogel preparation and other potential polysaccharides

The injectable LDN-193189 loaded hydrogel was prepared using k-carrageenan (1% w/v). The range can be 0.4 to 20% w/v of k-carrageenan. In brief, 7 mg of k-carrageenan solution was prepared in 665 pL of IX PBS by heating at 70°C until dissolution. Temperature can be varied from 45°C to 90°C. After dissolution, 35 pL of LDN-193189 (2 mg/mL in DMSO) was added to k-carrageenan solution, vortexed to make a homogenous solution followed by aspirating the whole solution in 30G syringe and left for overnight for gelation. The resulting hydrogel consists of 10 pg of LDN-193189 per 100 pL of hydrogel, similarly higher loading of LDN-193189 injectable hydrogels were prepared at 50 pg of drug per 100 pL of hydrogel. This kind of injectable hydrogel can also be made with other natural polymers such as alginic acid, chitosan, hyaluronic acid, collagen, gelatin and their combinations. Such polymer combination may induce ionic pair formation with the encapsulated drug.

Example 6: Drug release profile

The injectable hydrogels were evaluated for drug release profile using LCMS method. Both hydrogels (10 pg/100 pL and 50 pg/100 pL hydrogel) were loaded in dialysis membrane (3.5 KDa molecular weight cut-off) and outer chamber was filled with IX PBS. The hydrogel system was kept in an incubator shaker at 37°C with 50 rpm, the released dmg in outer chamber of dialysis membrane was collected at desired time point, lyophilized and reconstituted in lmL of methanol. The reconstituted methanol was centrifuged at 13000 rpm for 30 minutes at 4°C and supernatant was evaluated for LCMS quantification of the drug. The LCMS quantification results showed prolong sustained release of drug which was found to be 20-30% released in 6 days without any burst release. The release profile of the drug matches the requirement of drug dosage in in vivo experiment.

Example 7: in vitro activity of released drug using BRITER cell assay

To assess the activity of released dmg from the injectable hydrogel, in-house developed BMP Responsive Immortalized Reporter (BRITER) cell line were used. The BRITER cells are very specific and sensitive to exogenous BMP protein and leads to increase in expression of luciferase. Briefly, the BRITER cells were cultured and treated with BMP 50 ng/mL for 6 h , post 6 h the cells were washed with IX PBS and LDN-193189 (100 nM) was added as positive control (BMP inhibitor), only IX PBS (negative control), 400 pL of released LDN-193189 from injectable hydrogels (15 pg/100 pL of hydrogel) at 1-day, 3-day and 5-day. The results showed significant decrease in luciferase expression from the released drug till day-5 which was comparable with alone LDN-193189 (100 nM) treatment. Thus, the released drug from injectable hydrogels were stable upto day-5 and was in appropriate quantity which can inhibit the BMP signaling significantly.

Example 8:

Intra-articular injection of LDN-193189 loaded k-carrageenan hydrogel attenuates osteoarthritis:

To maintain slow and sustained release of LDN-193189 in joint cavity of mice for longer duration

The Inventors have used LDN-193189 loaded k-carrageenan hydrogel (1% w/v in PBS) having concentration of 500ng/pl. We have injected 6pl -8pl of LDN-193189 loaded k-carrageenan hydrogel in joint cavity at 1 and 2 months of post osteoarthritis induction in mice. Knee joints were harvested and articular cartilage were analyzed at 3 months of post-osteoarthritis. The articular cartilage markers were significantly maintained even after 2 months of drug administration as assessed by Colli and Col X immunoreactivity. The integrity of articular cartilage as evaluated by Safiranin O and Toluidine blue staining remain preserved even after 2 months of drug administration. The results are shown in Fig 11 (a) and (b).