Field of the Invention

The present invention relates to the treatment of bone fractures.

The invention particularly relates to the use of agomelatine, which is a melatonin receptor agonist, so as to increase/accelerate the healing of bone fractures.

Present State of the Art

Bone is the hardest of the tissues that make up the body. It is the tissue that serves as a support in the organism in real terms. They are also the calcium stores of the organism. They are hard because they are saturated with calcium. Although they are hard, their difference from cartilage tissue is that they contain vessels. Bone is among the rare organs in the human body that can repair itself.

Bone fracture is the deterioration of bone integrity due to direct or indirect trauma exceeding bone flexibility. It may develop anywhere in the bone. The main causes of fractures are: traumas such as traffic accidents, falling down, impact, buckling, domestic accidents, work accidents, sports accidents and injuries, objects falling on individuals, and gunshot injuries. The force which causes the fracture can not only break the bone, but also injure the soft tissues (skin, muscle, tendon, vein and nerves) around the bone. The condition of the surrounding tissues is very important when evaluating the fracture and determining the treatment and timing. The aim of treatment in bone fractures is to regain the function while maintaining body integrity. Mainly, bone fractures are treated using plaster or surgical fixation methods. Plate-screws, intramedullary nails, external fixators can be used as surgical fixation methods.

Fracture healing and duration differ from person to person. Age, gender, additional disorders (Diabetes, Dialysis, etc.), drugs used, smoking-alcohol use and nutrition are effective factors on fracture healing. For example, fracture healing takes an average of 4-8 weeks in childhood; on the other hand it takes an average of 5-12 weeks in adulthood.

The rate of surgery due to fracture has been increasing day by day in the last 20 years. The most important reason is the desire of people to return to their pre-fracture activity level earlier. For example, the patient was hospitalized for 6 months, 1 month in a sling due to a femur (thigh bone) fracture 30 years ago; it has been brought to a condition that can be expressed in days with the new method and implants used. Therefore, one of the most effective methods used to accelerate bone healing is the use of implants. There are many studies in the art on this subject.

In the Turkish Patent document numbered TR2018/14688 describes the cannulated telescopic screw used in the fixation of bone fractures is disclosed. This invention relates to is related to OTA 33B1-2 C1 -2-3, schatzker type 1 ,4 and 5 screws used in the fixation and healing of different types of bone fractures, patella transverse fractures, pubic and sacroiliac dissociation, characterized in that; it relates to cannuled telescopic screw (A) which is applied to the cavity (tunnel) opened in the bone by making mini cuts by matching the fracture lines in the broken bones, eliminates the unnecessary wound opening during the surgical intervention in this manner, consists of a female screw (10) and a male screw (20) in a telescopic structure, the attachment surface is increased and attached to the bone by means of the opposite external male screw (14) on the outer surface of the female screw (10), thus preventing the deformation of the bone during the fixing of the male screw (20), which is connected to the female screw path (11) in the female screw (10) in a way that is opposite the female screw (10) by the male screw path (21 ) on it, whose screw length is extended by reducing the connection distance of the male screw (20) to the female screw (10) provided that adequate compression pressure is maintained during the intervention. Although such implants accelerate the healing process, there is a risk of post-operative infection due to the placement of a foreign material in the body. Furthermore, patients with implants may experience problems over time. Also, insertion of implants requires painful and difficult surgeries. In addition to the discomfort that the patient will experience, these surgeries are costly processes.

There are also studies on the active substances that will accelerate the healing of bone fractures in the art. For example, European Patent document EP3164133B1 discloses compositions and products for use in the treatment of bone fractures and defects. The invention relates to methods and particulars method and compositions accelerating fracture healing, and injectable in situ deposition formulations for use in these methods comprising at least one biodegradable polymer, an androgen receptor agonist, and a bisphosphonate or acceptable salt thereof.

As a result, due to the abovementioned disadvantages and the insufficiency of the current solutions regarding the subject matter, a development is required to be made in the relevant technical field.

Aim of the Invention

The present invention relates to melatonin receptor agonist that fulfills the abovementioned requirements, eliminate all disadvantages and bring some additional advantages.

The main aim of the invention is to accelerate the fracture healing process, which is among the most common causes of job loss, and to shorten the fracture healing period.

Another aim of the invention is to reduce cost of fracture treatment.

The present invention relates to a melatonin receptor agonist suitable for use in enhancing/accelerating the healing of bone fractures, said melatonin receptor agonist is agomelatinin or a pharmaceutically acceptable salt thereof.

The structural and characteristic features and all avantages of the present invention will be understood clearly by the following detailed description. Therefore the evaluation shall be made by taking this detailed description into consideration.

Figures Clarifying the Invention

Figure 1A is radiography sample evaluated 21 days after K-wire application in an experimental fracture model in the rat

Figure 1 B is radiographic sample evaluated in the group treated with 30 mg/kg agomelatine for 21 days after K-wire application in an experimental fracture model created in the rat Figure 1 C is radiograph sample of 60 mg/kg agomelatine for 21 days after K-wire application in an experimental fracture model created in the rat

Figure 2 is radiographic fracture healing score graph

Figure 3 is RT-PCR ALP mRNA results

Figure 4 is RT-PCR OC mRNA results

Detailed Description of the Invention

In this detailed description, the preferred embodiments of the inventive melatonin receptor agonist are described only for clarifying the subject matter in a manner such that no limiting effect is created. The present invention relates to a melatonin receptor agonist suitable for use in enhancing/accelerating the healing of bone fractures, said melatonin receptor agonist is agomelatinin or a pharmaceutically acceptable salt thereof.

A formulation suitable for use is being developed to increase/accelerate the healing of bone fractures with the invention. The developed formulation mainly comprises; agomelatine as the melatonin receptor agonist or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of the invention, agomelatine or the formulation containing thereof is applied at dosages of 30 mg/kg or 60 mg/kg. Agomelatine in the ratios specified herein is preferably dissolved in 1 ml of distilled water and administered orally.

Agomelatine is an atypical antidepressant which is used for treating major depressive disorder. Agomelatine is an orally active, selective agonist for the melatonin MTNR1A and MTNR1 B receptors and a selective antagonist for the serotonin HTR2C receptor. The effect of agomelatine on fracture healing is revealed with the invention. The healing process of bone fracture, which has high treatment costs and is among the causes of job loss, is accelerated and fracture healing time is shortened with agomelatine.

40 male Wistar albino rats were randomly divided into four groups with 10 rats per group so as to understand the effect of the present invention. Rats are 10-12 weeks old and weigh 250 to 300 g. Animals were provided by Medical Experimental Research Center pf Ataturk University.

Group 1 (CONTROL or SHAM): Surgical Control Group

Group 2 (FRACTURE): Femur fracture group

Group 3 (KR+AGO30): Femur fracture + agomelatine 30 mg/kg (dissolved in 1 ml distilled water)

Group 4 (KR+AGO60): Femur fracture + agomelatine 60 mg/kg (dissolved in 1 ml distilled water)

During the operation, all rats are anesthetized intraperitoneally with 80 mg/kg of ketamine and 8 mg/kg of xylazine. The right rear extremity is shaved and then a 2 cm lateral femoral incision is made and the femur is reached. The femoral fracture model is created by cutting the middle section of the femur transversely. After manual reduction, the fractured femur is repaired with an intramedullary Kirschner wire. The wounds are reduced and closed using 4- 0 nylon sutures, and the soft tissue and skin are closed with 4-0 Vicryl sutures. A peritoneal injection of metamizole sodium (150 mg/kg) is provided at the end of the surgical procedure so as to prevent post-operative pain. One day after surgery, rats are anesthetized by peritoneal injection of 80 mg/kg of ketamine and 8 mg/kg of xylazine and subjected to X-ray imaging. Rats are allowed to drink and eat freely. Two different doses of agomelatine are administered and control rats are given the same volume of water instead of this. Rats are given 30 or 60 mg/kg agomelatine orally (by gavage) once a day for 21 days, starting on postoperative day 1. Twenty-one days after surgery, rats are anesthetized by peritoneal injection of 80 mg/kg of ketamine and 8 mg/kg of xylazine, after imaging, anesthetized with ketamine and xylazine again and euthanized for tissue collection. Femurs are stored at -80 O for molecular analysis and fixed in 10% formalin for histopathological analysis. Bonespecific alkaline phosphatase (ALP), osteocalcin (OC) and osteopontin (OP), which are markers of healing in bone tissues, are molecularly evaluated by Real Time Polymerase Chain Reaction (RT-PCR) and immunohistochemistry staining. Fracture healing is evaluated by hematoxylin-eosin staining in tissues histopathologically. RT-PCR is used to evaluate bone ALP, OC and OP mRNA expression levels. Therefore, homogenization of bone tissues, RNA isolation, cDNA synthesis and quantitative determination of mRNA expressions are performed. Fracture healing is evaluated by hematoxylin-eosin staining in tissues histopathologically. Kruskal Wallis (with Bonferroni correction) is used as a statistical method in the evaluation of the data.

The results obtained from the x-rays are given in Figure 1A, 1 B and 1 C. Accordingly, Figure 1A gives a radiographic example evaluated 21 days after K-wire application in a experimental fracture model created in the rat, Figure 1 B gives a radiographic example evaluated in the group treated with 30 mg/kg agomelatine for 21 days after K wire application in an experimental fracture model created in rat and Figure 1C gives a radiographic example evaluated in the experimental fracture model created in the rat, in the group treated with 60 mg/kg agomelatine for 21 days after the K-wire application. The results obtained from the x- rays are scored as follows:

1 : Pseudoarthrosis

2: Incomplete cartilaginous union

3: Complete cartilaginous union

4: Incomplete bony union

5: Complete bony union X-ray images of femur fractures are evaluated at 21 days so as to determine the degree of fracture healing. Fracture healing stage is assessed using a modified five-point radiographic scoring system. An investigator who is blind to treatment groups is evaluating radiographs for X-ray analysis.

Radiographic fracture healing score graph is given in Figure-2, thus a statistically significant increase was observed in the KR+ AGO60 (4.2 ± 0.44721 , p=0.041) group compared to the FRACTURE group (3.4 ± 0.54772) (p<0.05). KR fracture in Figure-2, AGO 30 means: 30 mg/kg agomelatine, AGO 60: 60 mg/kg agomelatine.

Ribonucleic acid (RNA) extraction, reverse transcriptase reaction and complementary DNA (cDNA) synthesis and quantitative determination of OC, OP and ALP mRNA expressions by RT-PCR are performed from rat femoral tissue for Real-Time PCR. In the ribonucleic acid (RNA) extraction from rat femoral tissue; tissues (20 mg) are homogenized using TissueLyser II (Qiagen) with nitrogen treatment; RNA extraction is performed in the QIAcube and total RNA isolation is performed. The total amount of mRNA is measured by nanodrop spectrophotometry (EPOCH Take 3 Plate, Biotek) at 260 nm. The obtained RNA is stored at -80 O under the required conditions.

In reverse transcriptase reaction and complementary DNA (cDNA) synthesis, cDNA production from RNA is performed with the High Capacity cDNA Reverse Transcription Kit. 10 pl of RNA is used in each reaction according to the temperature values in the Veriti 96 Well Thermal Cycle (Applied Biosystem) for cDNA synthesis. The amount of cDNA was determined by nanodrop spectrophotometry (EPOCH Take3 Plate, Biotek) and the obtained cDNA is stored at -20 O. A total of 10 pl of RNA, 2 pl of 10 x RT Buffer, 0.8 pl of 25 x dNTP mix, 2 pl of 10 x RT random primer, 1 MultiScribe Reverse Transcriptase, and 4.2 pl of diethylprocarbonate H20 are used for the DNA synthesis reaction.

Quantitative determination of OC, OP and ALP mRNA expressions by RT-PCR, OC, OP and bone ALP mRNA expression is achieved using TaqMan® Gene Expression Master Mix. B- actin is used as the reference gene. Amplification and quantification are performed using the StepOne Plus RT-PCR system (Applied Biosystems). Subsequent TaqMan gene expression analyzes for 200 ng of cDNA are performed by pipetting over 40 cycles as described below. The cycle threshold (CT) is the number of cycles in which the amount of fluorescent signal in real-time PCR experiments exceeds the minimum value (threshold) required to be observed. CT values were automatically converted to AACt (Livak and Schmittgen, 2001) and the results were statistically evaluated in the Statistical Package for Social Sciences (SPSS) 25.0. X pl of cDNA (200 ng), 10 pl of TaqMan Master Mix and 1 pl of assay were used for pipetting and filled to 20 pl with RNase-free HO.

It was observed that also, ALP mRNA expressions in KR+AGO 30 (1 ,039 ± 0,007, p=0,045) and KR+AGO60 (1 ,390 ± 0,007, p<0,01 ) groups increased statistically and significantly (p<0.05) compared to the FRACTURE (0,769 ± 0,015) group. The graph of RT-PCR ALP mRNA expressions is given in Figure 3. It was observed that also, OC mRNA expressions in KR+AGO 30 (1.418 ± 0.005, p=0.000) and KR+AGO60 (1.190 ± 0.007, p=0.044) groups increased statistically and significantly (p<0.05) compared to the FRACTURE (1 ,039± 0.007) group. The graph of RT-PCR OC mRNA expressions is given in Figure 4. Histopathological results also support radiographic and molecular results. Herein, scoring is performed similar to previous studies, based on the presence of phases (inflammatory cells, fibrous tissue, cartilage tissue, braided bone tissue) observed in bone healing and the surface areas they cover so as to make the histopathological findings more clear. Scoring was made based on the rareness and excess of the involvement regions and is presented in Table 1 below so as to make the immunohistochemical staining findings more clear. In the table, KR: It means; Fracture, AGO: agomelatin, Grade 0:- (0% absent), Grade 1 : +(0-33% mild), Grade 2:++ (33- 66% moderate), Grade 3: +++ (66-100% severe).

Table-1

Histopathological analyses were also performed so as to understand the effect of the invention. Histological and hematoxylin-eosin procedure are as follows.

Samples of hard bone tissue in the fixative are dipped in 10% nitric acid. The solution is changed daily and tissue softening degree is determined by a needle. Decalcification occurs in tissues for 2-10 days. Then, the tissues are washed overnight and then dehydration, xylol clearing, and paraffin infiltration are performed on alcohol blocks. The following steps are performed with a Leica TP 1050 instrument: 10% buffered formalin (1 hour, 15 minutes), 70% alcohol (1 hour), 80% alcohol (1 hour), 90% alcohol (1 hour), pure alcohol (1 hour), pure alcohol (1 hour), pure alcohol (1 hour, 15 minutes), xylol (1 hour), xylol (1 hour), xylol (1 hour, 15 minutes), paraffin (1 hour), paraffin (1 hour), and paraffin (1 hour, 15 minutes)). The device is operated at 40 'C in formalin, alcohol an d xylol stages and at 65 'C in paraffin stages. Tissues obtained after monitoring are blocked with paraffin melted at 67 G in a Leica EG1160 blocking device, and the blocked tissues are cooled at -4 6. A 5 micron thick section is taken with a Leica RM 2145 microtome.

As a result of these analyzes, histopathology results are obtained. Here, hematoxylin and eosin staining results are examined. The following results are revealed in histopathological examinations with HE staining in the femoral tissues of rats in all groups:

CONTROL (SHAM): When the longitudinal section of the femur bone is examined, there is no anomaly in the general appearance of the compact bone, the dimensions and location of the bone canals, the organic content of the extracellular matrix and the cell integrity. While the spongiform bone integrity was normal in other groups; spongiform bone spicules are observed in this group.

FRACTURE: When the image is examined, cartilage tissue formation is observed at the level of the spongiform bone in the transverse fracture line; it is determined that the broken ends do not come together during the treatment in the compact bone line. Furthermore, inflammatory cells are observed in the compact bone line, indicating that the inflammation phase continues.

FRACTURE + AGO 30 mg/kg: Intense cartilage tissue deposition is observed at the fracture line. While chondrocytes have mostly normal appearance, some of them have developed into hypertrophic state. Hard callus and braided bone are detected near the spongiform bone. Healing lines with conic-ends that are located perpendicular to the fracture line and extend from one ossification region to the other are observed.

FRACTURE + AGO 60 mg/kg: It was observed that the cartilage tissue accumulation was considerably reduced compared to the FRACTURE + AGO 30 mg/kg group, instead the braided bone is dominant. It has been analyzed that the braided bone formed at the level of the spongiform bone also has a hollow appearance. Fibrous tissue residuals are observed in the area close to the periosteum.

No involvement was observed in the fracture group during the staining performed with antibodies interacting with osteocalcin protein; involvement was observed in the cells of the cartilage tissue adjacent to the spongiform bone and the extracellular matrix in the treatment group. Herein, the involvement observed in the AGO groups is directly proportional to the dose increase.

When the immunohistochemistry staining findings of the osteopontin group were examined, involvement was observed in all treatment groups including the fracture group. However, there are no significant differences in the amount of involvement between the groups. The staining regions are concentrated in the extracellular matrix compared to the cell. When alkaline phosphatase staining is examined, staining regions are observed as focal in the fracture and AGO groups.

It is shown radiologically, molecularly and histopathologically that agomelatine increases bone fracture healing with the invention. While accelerating the fracture union process with the invention, simultaneously, it is ensured that the symptoms of psychological illness that occur during the fracture healing process are eliminated, and the financial burden of the patient is eliminated.