Composition for the treatment of a disease of the musculoskeletal system in a dog

Description

Scope of the invention

[0001] The present invention relates to a composition useful for the prevention or treatment of a disorder of the musculoskeletal system. Technical background

[0002] In France, the total number of horses is approximately 350,000, of which roughly 28,000 are racehorses. Between 20 and 30% of these horses suffer from tendinopathies. This represents about 8,000 cases per year, considering only horse racing.

[0003] No conventional treatment method based on anti-inflammatories offers any real benefit compared to natural, long-term healing, which requires rest for six months to a year. Furthermore, these treatments are primarily symptomatic and do not heal the tendon.

[0004] It has therefore been proposed to use stem cells, particularly from bone marrow, umbilical cord or adipose tissue, in order to obtain tendon healing that closely resembles the original tendon.

[0005] However, adipose tissue cells appear to differentiate less well than bone marrow cells. Furthermore, the reliability of umbilical cord blood stem cells is not yet fully established.

[0006] Thus, mesenchymal stem cells from bone marrow are currently considered the reference treatment for equine tendinitis (Godwin et al. (2012) Equine Veterinary Journal 44:25-32).

[0007] However, bone marrow harvesting is a relatively complex procedure. Furthermore, mesenchymal stem cells from bone marrow have primarily been validated for autologous use, meaning the cells originate from the animal to which they are administered, which increases the cost of this method.

[0008] It would therefore be beneficial to have alternative treatments that are simpler and less expensive to implement. Summary of the invention

[0009] The present invention arises from the unexpected discovery by the inventors that administering a composition based on heterologous gingival fibroblasts to horses suffering from tendonitis or osteochondrosis made it possible to treat these conditions.

[0010] Advantageously, gingival fibroblasts are easily harvested and prepared. Furthermore, they are effective even in heterologous applications. The invention is as defined in the claims. Detailed description of the invention

[0011] As a preliminary point, it should be noted that the term "comprising" means "including," "containing," or "encompassing," meaning that when an object "comprising" one or more elements, elements other than those mentioned may also be included within the object. Conversely, the expression "consisting of" means "made up of," meaning that when an object "consists of" one or more elements, the object cannot include elements other than those mentioned. Musculoskeletal disorder

[0012] As understood here, a locomotor system disorder refers in particular to an osteoarticular or musculoskeletal disorder, especially of a limb.

[0013] Diseases of the horse's locomotor system are described in particular in "Horse Disease" (1994) Institut du cheval, France Agricole Editions.

[0014] The condition affecting the locomotor system according to the invention is tendinitis.

[0015] More preferably, the locomotor system disorder according to the invention is a tendinitis of a flexor tendon, in particular superficial or deep of the phalanges, or of an extensor tendon, in particular lateral of the phalanges or anterior of the phalanges, particularly in a horse.

[0016] As we understand it here, the superficial flexor tendon is also called the perforated tendon and the deep tendon of the phalanges is also called the perforating tendon.

[0017] Alternatively, the locomotor system disorder according to the invention is osteochondrosis or osteoarthritis.

[0018] As we understand it here, osteoarthritis is synonymous with osteoarthritis.

[0019] Preferably, osteochondrosis or osteoarthritis is associated with bone nodules, osteochondritis dissecans, or a bone cyst. Individual

[0020] The individual according to the invention is a The individual according to the invention can be male or female. Composition

[0021] As understood here, the expression "buccal cells" is equivalent to "oral cavity cells" or "oral mucosal cells".

[0022] The buccal cells according to the invention are gum cells.

[0023] Preferably, the composition according to the invention is obtained by culturing a gum sample.

[0024] The composition according to the invention comprises or is composed of gingival fibroblasts. This is then referred to as a gingival fibroblast composition. The composition according to the invention may, in particular, be a culture, possibly concentrated, of gingival fibroblasts or a suspension of gingival fibroblasts.

[0025] The procedures for collecting, culturing, and preserving gingival fibroblasts are well known to those skilled in the art and are described in particular in Naveau et al. (2006) J. Periodontol. 77:238-47 and in Gogly et al. (2007) Arterioscler. Thromb. Vasc. Biol. 27:1984-90, as well as in the examples below. Specifically, gingival fibroblasts can be obtained by culturing a gingival tissue sample or biopsy, possibly after enzymatic digestion of the sample or biopsy to release the gingival fibroblasts that constitute it. Thus, the cells obtained from culturing a gingival tissue sample or biopsy are essentially gingival fibroblasts.

[0026] In one embodiment of the invention, the gingival fibroblasts according to the invention comprise mesenchymal stem cells. As those skilled in the art understand, the mesenchymal stem cells are then buccal mesenchymal stem cells, that is to say, from the oral cavity or the oral mucosa, in particular the mucosa of the cheek, tongue, palate, labial mucosa, sublingual mucosa, or gingiva.

[0027] In another embodiment of the invention, the gingival fibroblasts according to the invention do not include, or essentially do not include, mesenchymal stem cells.

[0028] Advantageously, gingival fibroblasts are easily harvested and cultured. Furthermore, gingival fibroblasts exhibit a high growth rate.

[0029] As those skilled in the art will readily understand, the composition according to the invention comprises gingival fibroblasts according to the invention in a prophylactically or therapeutically effective quantity. Preferably, the composition according to the invention comprises from 5 million to 40 million gingival fibroblasts according to the invention, more preferably from 10 million to 30 million gingival fibroblasts according to the invention, even more preferably from 15 million to 25 million gingival fibroblasts according to the invention, and most preferably about 20 million gingival fibroblasts according to the invention. The quantification of the gingival fibroblasts according to the invention can be performed by any method of counting mammalian cells known to those skilled in the art, and in particular using an automated cell counter.

[0030] Preferably, the gingival fibroblasts according to the invention are heterologous, that is, they originate from a different individual, in particular a different horse, than the one in which the composition is used. As those skilled in the art will readily understand, the individual from which the cells are harvested and the individual to which the cells are administered are preferably of the same species.

[0031] Preferably also, the gingival fibroblasts according to the invention are autologous, that is to say, they come from the same individual, in particular the same horse, as the one in which the composition is used.

[0032] Preferably, the composition according to the invention is administered near or at the site of treatment, more preferably into the lesion to be treated. Administration can be carried out by any method known to those skilled in the art that is compatible with cell administration. However, it is preferable that the composition be injected, particularly into the lesion to be treated. Preferably, the composition according to the invention is thus injected at the site of tendinitis to be treated, more preferably into the tendon lesion, notably by ultrasound guidance, or into or at the site of a joint exhibiting osteochondrosis or osteoarthritis.

[0033] The invention will be further explained in a non-limiting manner by the Figure and Examples that follow. Description of the figure

[0034] Figure 1 shows ultrasound scans of the superficial flexor tendon of 4 horses suffering from tendinitis of this tendon before injection of a composition of gingival fibroblasts (Day 0) and then 3 months after injection (Day 0 + 3 months).

[0035] The area with the tendon lesion, darker than its surroundings, is circled in white. The solution is injected into the lesion using a syringe (arrow). Three months later, the tendon has a smooth appearance, indicating that the lesion has been treated. EXAMPLES (These examples are not within the scope of the claims, but are included for reference purposes only.) Example 1 : treatment for tendinitis Preparation of gingival fibroblasts for administration

[0036] A biopsy of approximately 50 mg of gingiva is performed with a scalpel on a horse (horse 0) and then enzymatically digested to yield a suspension of gingival fibroblasts. After counting the suspension, a 75 cm² flask is inoculated with 3 x 10⁵ gingival fibroblasts in complete culture medium (DMEM 4.5 g / L glucose + Glutamax (Gibco®), fetal bovine serum (FBS) (Gibco®) 20%, in the presence of antibiotics / antifungals) and is incubated at 37°C in a 5% CO₂ atmosphere until 90% confluence is reached, with the culture medium being changed regularly.

[0037] Gingival fibroblasts are then collected. 5 x 10⁵ gingival fibroblasts are used to inoculate 25 mL of complete medium in a 150 cm² flask. Incubation is then carried out at 37°C under a 5% CO₂ atmosphere, with the culture medium being changed regularly, until 90% confluence is reached. This constitutes the first passage.

[0038] Two further passes are performed. During the third pass, approximately 20 x 10⁶ gingival fibroblasts are collected, centrifuged, washed in 10 mL of DMEM 0% SVF medium without antibiotics / antifungals, centrifuged again, and collected in a volume of approximately 3 mL. The gingival fibroblasts are then aspirated into the administration syringe and stored at 4°C until administration. Gingival fibroblast administration

[0039] Four horses (horses 1, 2, 3, and 4) presenting with tendinitis of a superficial flexor tendon, possibly associated with lameness, received an injection of heterologous gingival fibroblasts prepared as described above. The injured area of ​​the tendon was identified by ultrasound (see the Figure It appears as a denser (darker) area compared to its surroundings.

[0040] The injection of approximately 20.10 6< gingival fibroblasts is performed directly into the lesion by ultrasound guidance (intralesional injection). Results

[0041] Three months after the injection, an ultrasound examination of the treated area is performed (see the Figure There is no longer any apparent tendon damage: the treated area appears homogeneous on ultrasound. The animals' locomotion is normal. Example 2: Treatment of osteochondrosis

[0042] Gingival fibroblasts prepared as described in the Example 1 are injected directly into the fetlock joint of a horse (horse 5) with osteochondrosis of the fetlock associated with lameness.

[0043] An absence of lameness is observed approximately one week after the injection.

Claims

1. A composition comprising gingival fibroblasts for use in the prevention or treatment of a disease of the locomotor system in a dog selected in the group consisting of a tendinitis, an osteochondrosis and an arthrosis.

2. The composition for use according to claim 1, in the prevention or treatment of a tendinitis of a flexor tendon, in particular superficial or deep of the phalanges, or of an extensor tendon, in particular lateral of the phalanges or anterior of the phalanges.

3. The composition for use according to claim 1 or 2, wherein the composition is derived from the culturing of a gum sample.

4. The composition for use according to any one of claims 1 to 3, wherein the composition comprises from 5 million to 40 million gingival fibroblasts.

5. The composition for use according to any one of claims 1 to 4, wherein the gingival fibroblasts are heterologous, which means they are taken from another dog than the one in whom the composition is used.

6. The composition for use according to any one of claims 1 to 4, wherein the gingival fibroblasts are autologous, which means they are taken from the same dog than the one in whom the composition is used.