PHYTOECDYSONES FOR USE IN THE PREVENTION OF MUSCLE STRENGTH LOSS DURING IMMOBILISATION

Abstract

A composition including at least one phytoecdysone or at least one semi-synthetic derivative of a phytoecdysone, for use in mammals for preventing the loss of muscle strength during immobilisation. More particularly, a composition for use that includes 20-hydroxyecdysone or a semi-synthetic derivative of 20-hydroxyecdysone. Moreover, the composition for use includes a compound of general formula (I).

Claims

1. A method for preventing loss of muscle strength during immobilization in mammals comprising administering to a mammal a pharmaceutically acceptable formulation suitable for oral administration including at least one phytoecdysone or at least one semisynthetic derivative of a phytoecdysone.

2. The method according to claim 1, wherein the formulation includes 20-hydroxyecdysone or a semisynthetic derivative of 20-hydroxyecdysone.

3. The method according to claim 1, wherein the formulation includes a compound of general formula (I): ##STR00004## wherein: R1 is chosen from: a (C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)W(C1-C6) group; a (C1-C6)W(C1-C6)CO2(C1-C6) group; a (C1-C6)A group, A representing a heterocycle, optionally substituted by a group chosen from OH, OMe, (C1-C6), N(C1-C6), CO2(C1-C6); a CH2Br group; W being a heteroatom chosen from N, O and S.

4. The method according to claim 1, wherein the formulation includes a compound chosen from the following compounds: n° 1: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one, n° 2: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(3-hydroxypyrrolidin-1-ypacetyl]-10,13- dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; n° 3: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-1742-(4-hydroxy-1-piperidyl[acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; n° 4: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-[4-(2-hydroxyethyl)-1-piperidyl]acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one; n° 5: (2S,3R,5R,10R,13R,14S,17S)-17-[2-(3-dimethylaminopropyl(methyl)amino)acetyl]-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1 H-cyclopenta[a]phenanthren-6-one; n° 6: ethyl 2-[2-oxo-2-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]sulfanylacetate; n° 7: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethylsulfanylacetyl)-2,3,14-trihydroxy10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1 H-cyclopenta[a]phenanthren-6-one; n° 8: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethyl sulfanyl)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H cyclopenta[a]phenanthren-6-one.

5. The method according to claim 1, wherein the formulation includes a compound of formula (II): ##STR00005##

6. The method according to claim 1, wherein the phytoecdysones are administered at a dose of between 50 and 1000 milligrams per day in humans.

7. The method according to claim 1, wherein the formulation is administered during immobilisation.

8. The method according to claim 1, wherein the formulation is administered until immobilisation ends.

9. The method according to claim 7, wherein the formulation is also administered during a predetermined period after ending of immobilisation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Other particular advantages, aims and features of the present disclosure will emerge from the following non-limitative description of at least one particular aspect of the object of the present disclosure, with regard to the accompanying drawings, wherein:

[0067] FIG. 1A is an image representing histological sections coloured with haematoxylin and eosin of anterior tibial (AT) muscle of a mouse of genetic background C57BL/6J non-immobilised,

[0068] FIG. 1B is an image representing histological sections coloured with haematoxylin and eosin of anterior tibial (AT) muscle of a mouse of genetic background C57BL/6J immobilised and treated with the vehicle for 14 days,

[0069] FIG. 1C is an image representing histological sections coloured with haematoxylin and eosin of anterior tibial (AT) muscle of a mouse of genetic background C57BL/6J immobilised and treated with the compound of formula (II) for 14 days.

[0070] FIG. 1D is a diagram representing the area of the muscle fibres of the anterior tibial muscle of a mouse of genetic background C57BL/6J non-immobilised (control), immobilised and treated with the vehicle for 14 days or immobilised and treated with a compound of formula (II) for 14 days,

[0071] FIG. 2A is a diagram representing the weight of the anterior tibial muscle of groups of mice of genetic background C57BL/6J non-immobilised (control), immobilised and treated with the vehicle for 14 days or immobilised and treated with the compound of formula (II) for 14 days.

[0072] FIG. 2B is a diagram representing the weight of the gastrocnemius muscle of groups of mice of genetic background C57BL/6J non-immobilised (control), immobilised and treated with the vehicle for 14 days or immobilised and treated with the compound of formula (II) for 14 days.

[0073] FIG. 3A depicts the absolute maximum isometric force of the anterior tibial muscle of a mouse of generic background C57BL/6J at various times post-immobilisation: non-immobilised (J0), after 14 days of immobilisation (J14), after 14 days of immobilisation and 1 week of remobilisation (J21) or after 14 days of immobilisation and 2 weeks of remobilisation (J28), treated with the vehicle or with the compound of formula (II).

[0074] FIG. 3B depicts the specific maximum isometric force of the anterior tibial muscle of a mouse of genetic background C57BL/6J at various times post-immobilisation: non-immobilised (J0), after 14 days of immobilisation (J14), after 14 days of immobilisation and 1 week of remobilisation (J21) or after 14 days of immobilisation and 2 weeks of remobilisation (J28), treated with the vehicle or with the compound of formula (II).

[0075] FIG. 4A is a diagram representing the weight of the anterior tibial muscle of groups of mice of genetic background C57BL/6J non-immobilised (control, measured at JO on non-immobilised animals), immobilised and treated with the vehicle for 14 days or immobilised and treated with the compound BIO101 for 14 days.

[0076] FIG. 4B is a diagram representing the weight of the gastrocnemius muscle of groups of mice of genetic background C57BL/6J non-immobilised (control), immobilised and treated with the vehicle for 14 days or immobilised and treated with the compound BIO101 for 14 days.

[0077] FIG. 5A is a diagram depicting the absolute maximum isometric force of the anterior tibial muscle of a mouse of genetic background C57BL/6J non-immobilised (control, measured at J0 on non-immobilised animals), immobilised and treated with the vehicle for 7 days or immobilised and treated with the compound BIO101 for 7 days.

[0078] FIG. 5B is a diagram depicting the specific maximum isometric force of the anterior tibial muscle of a mouse of genetic background C57BL/6J non-immobilised (control, measured at J0 on non-immobilised animals), immobilised and treated with the vehicle for 7 days or immobilised and treated with the compound BIO101 for 7 days.

[0079] FIG. 6A depicts the absolute maximum isometric force of the anterior tibial muscle of a mouse of genetic background C57BL/6J at various times post-immobilisation: non-immobilised (JO), after 7 days of immobilisation (J7), after 14 days of immobilisation (J14), and after 14 days of immobilisation and then 2 weeks of remobilisation (J28), treated with the vehicle or with the compound BIO101.

[0080] FIG. 6B depicts the specific maximum isometric force of the anterior tibial muscle of a mouse of genetic background C57BL/6J at various times post-immobilisation: non-immobilised (J0), after 7 days of immobilisation (J7), after 14 days of immobilisation (J14), and after 14 days of immobilisation and then 2 weeks of remobilisation (J28), treated with the vehicle or with the compound BIO101.

DETAILED DESCRIPTION

Method for Synthesising the Compound of Formula (II)

[0081] The compound of formula (II) to which reference is made in the rest of the description is as follows:

##STR00003##

[0082] The compound of formula (II) is obtained by semisynthesis from 20-hydroxyecdysone and then purification to pharmaceutical grade.

[0083] The method for preparing the compound of formula (II) by semisynthesis includes in particular: [0084] a step of oxidising cutting of the side chain of the 20-hydroxyecdysone between carbons C20 and C22 in order to obtain poststerone, [0085] a step of introducing a bromine atom at position C21, and [0086] a step of reacting the bromine derivative with ethanethiol.

Biological Activity of the Compound of Formula (II)

[0087] A model of immobilisation of a posterior paw of a mouse of genetic background C57BL/6J was implemented by means of a tube (Lang et al., 2012).

[0088] Female C57BL/6J mice aged 13 weeks were used. Ten mice were sacrificed at J0, these mice were not immobilised in order to serve as a control.

[0089] J0, J14, J21, J28 means the time elapsed as from the start of the experiment, expressed in days. Thus J0 designates the start of the experiment (before treatment and before immobilisation), J14 designates the 14th day as from the start of the experiment, etc.

[0090] Two groups of mice were formed, a test group and a reference group. Each group is exposed, orally, chronically either to the vehicle (reference group) or to the compound of formula (II) at a dose of 50 mg/kg per day (test group). The oral treatment over 28 days consists of tube feeding for five days per week and in drinking water for two days per week.

[0091] The animals in all the groups were tested for their functional capacity in situ by means of measurements of the absolute and specific maximum isometric force of the anterior tibial (AT) muscle (FIGS. 3A and 3B) after 14 days of immobilisation (n=13 for the vehicle, n=10 for the compound of formula (II)), after 14 days of immobilisation and one week of remobilisation (n=7 for the vehicle, n=8 for the compound of formula (II)) and after 14 days of immobilisation and two weeks of remobilisation (n=6 for the vehicle, n=8 for the compound of formula (II)).

Histology and Atrophy of the Muscles (FIG. 1)

[0092] A histological study of the anterior tibial muscle is carried out on sections coloured with haematoxylin and eosin (HE). The area of the muscle fibres is evaluated on control-mouse muscles, or treated with the vehicle or with the compound of formula (II). The muscle in all cases presents a histology of healthy muscle tissue (FIGS. 1A to C); on the other hand, as might be expected, after 14 days of immobilisation, the mean area of the fibres is greatly reduced in animals that received the vehicle compared with the control animals (−24.4%, p=0.006) that have not been immobilised. The area of the muscle fibres of the group treated with the compound of formula (II) is also reduced compared with the control group (−26.8%, p=0.002).

[0093] No significant difference is therefore observed between the groups of animals treated with the vehicle and the group treated with compound of formula (II) (p=0.73). After 14 days of immobilisation, the compound of formula (II) therefore does not exert any protective effect against loss of muscle volume.

Weight of the Anterior Tibial (AT) and Gastrocnemius Muscles (FIG. 2)

[0094] The weight of the AT muscles (FIG. 2A) and gastrocnemius muscles (FIG. 2B) were evaluated in non-immobilised (control) mice, and after 14 days of immobilisation in mice treated either with the vehicle or with the compound of formula (II), during the 14 days of immobilisation.

[0095] As expected, it is observed that immobilisation causes a reduction in the muscle mass of the AT and of the gastrocnemius in mice that received the vehicle compared with the control group (−34.9%, p<0.001 and −29%, p<0.001 respectively).

[0096] It is observed that the weight of the AT and gastrocnemius muscles does not vary significantly in the group of mice treated with the compound of formula (II) compared with the vehicle. Consistent with the results obtained on the diameter of the muscle fibres (FIG. 1), the compound of formula (II) therefore does not exert any protective effect against the loss of muscle mass following an immobilisation.

Absolute and Specific Maximum Isometric Force of the Anterior Tibial Muscle (in situ Functional Study (FIG. 3))

[0097] An evaluation of the in situ contractility of the AT muscle is carried out at different times in the protocol: on non-immobilised control mice (J0), on mice subjected to an immobilisation of the posterior paw for 14 days (J14), immobilised for 14 days and then remobilised for 1 week (J21) and immobilised for 14 days and then remobilised for 2 weeks (J28).

[0098] On the day of sacrifice, the mouse is anaesthetised with an intraperitoneal injection of pentobarbital (55 mg/kg, 0.1 ml/10 g of body weight) before measuring the in situ force of the anterior tibial (AT) muscle. The skin on the top of the paw is incised, which reveals the tendon, which is cut at the distal end thereof. The distal tendon of the AT is attached to the lever of the servomotor (305B Dual-Mode Lever, Aurora Scientific). The skin on the lateral face of the thigh is incised, which reveals the sciatic nerve, between two muscle groups. The sciatic nerve is stimulated with a bipolar electrode (supramaximal pulse with a square wave of 10 V, 0.1 ms). The force is measured during contractions in response to the electrical stimulation (frequency of 75-150 Hz, duration 500 ms). The temperature of the mouse is maintained at 37° C. by means of a radiant lamp. The absolute maximum isometric force is measured (FIG. 3A) and the specific maximum isometric force (FIG. 3B) is calculated by comparing the absolute isometric force with the weight of the anterior tibial muscle.

[0099] As expected, it is found that the animals treated with the vehicle have an absolute maximum isometric contraction force significantly less than that of the non-immobilised control animals (−65.6%, p<0.001) (FIG. 3A). The animals treated with the compound of formula (II) exhibit a lesser absolute force loss (−26.9%, p=0.015) compared with the control, than the animals treated with the vehicle.

[0100] Surprisingly, it is observed that the treatment with the compound of formula (II) enables the animals immobilised for 14 days to preserve an absolute isometric force that is significantly greater than the animals treated with the vehicle and improves their performance (+112.1%, p=0.0041). This despite the absence of any effect of the compound of formula (II) on the loss of mass and muscle volume noted previously.

[0101] It is observed that the animals treated with the vehicle have a specific maximum isometric contraction force (sP0; FIG. 3B) significantly less than that of the non-immobilised control animals (−57.8%, p<0.001). Remarkably, the specific force of the animals treated with the compound of formula (II) is not significantly affected by the immobilisation: −8% (p=0.32) compared with the animals in the control group that were not immobilised. The treatment with the compound of formula (II) enables the animals immobilised for 14 days to preserve a normal muscle function while doubling the specific isometric force (+117.6%, p<0.001) compared with the animals in the immobilised group, treated with the vehicle.

Biological Activity of the Compound BIO101

[0102] A second study was carried out using the same method of immobilisation of the posterior paws, on mice with the same age (13 weeks) and the same genetic background (C57BL/6J) as described previously, but adding an analysis point after 7 days of immobilisation. The analysis points are therefore J0, J7, J14 and J28.

[0103] Ten mice were sacrificed at J0, these mice were not immobilised in order to serve as controls (control group in the Figures).

[0104] J7, J14, J28 means the time elapsed as from the start of the experiment, expressed in days. Thus J7 designates the 7.sup.th day as from the start of the experiment, etc.

[0105] Two groups of mice were formed, a test group and a reference group. Each group is exposed, orally, chronically either to the vehicle (reference group) or to the compound BIO101 at a dose of 50 mg/kg per day (test group). Compound BIO101 means a plant extract, said plant being chosen from plants containing at least 0.5% 20-hydroxyecdysone by dry weight of said plant, said extract including by way of active agent 20-hydroxyecdysone in a quantity of at least 95%, and preferably at least 97% by weight with respect to the total weight of the extract. The oral treatment for 28 days consists of tube feeding for five days per week and administration in drinking water for two days per week.

[0106] The animals in all the groups were tested for their functional capacity in situ (the two posterior paws) by means of measurements of the absolute and specific maximum isometric force of the anterior tibial (AT) muscle (FIGS. 6A and 6B) after 7 days of immobilisation (n=6 mice, two values per mouse for the reference group (vehicle), n=6 mice, two values per mouse for the test group (BIO101), after 14 days of immobilisation followed by two weeks of remobilisation (n=6 per mouse, two values per mouse for the vehicle (reference group), n=6 mice, two values per mouse for the compound BIO101), and after 14 days of immobilisation followed by two weeks of remobilisation (n=6 per mouse, two values per mouse for the vehicle (reference group), n=6 mice, two values per mouse for the compound BIO101).

Weight of the Anterior Tibial (AT) and Gastrocnemius Muscles (FIG. 4)

[0107] The weight of the AT (FIG. 4A) and gastrocnemius (FIG. 4B) muscles were evaluated in non-immobilised mice (control group), and after 14 days of immobilisation in mice treated either by the vehicle or by the compound BIO101 throughout the duration of immobilisation. As expected, it is observed that the immobilisation causes a reduction in the muscle mass of the AT (−21.7%, p<0.001) in mice that received the vehicle compared with a control group, non-immobilised (FIG. 4A).

[0108] It is observed that the weight of the AT and gastrocnemius muscles does not vary significantly in the test group of the mice treated with the compound BIO101 compared with the vehicle-treated reference group (FIGS. 4A and 4B).

Absolute and Specific Maximum Isometric Force of the Anterior Tibial Muscle (in situ Functional Study (FIGS. 5 and 6))

[0109] An evaluation of the contractility in situ of the AT muscle is made at various times in the protocol: on non-immobilised control mice (control group, J0), on mice subjected to immobilisation of the posterior paws for 7 days (J7), 14 days (J14), immobilised for 14 days and then remobilised for 2 weeks (J28).

[0110] When the force developed by the AT muscle is considered after seven days of immobilisation, as expected it is found that the animals treated with the vehicle (reference group) have an absolute maximum isometric contraction force significantly less than that of the non-immobilised control animals (−34.7%, p<0.001) (FIG. 5A). The animals treated with the compound BIO101 exhibit a loss of absolute force that is less (−21.1%, p=0.001) compared with the control, than the animals treated with the vehicle (FIG. 5A).

[0111] Interestingly, it is observed that the treatment with the compound BIO101 enables the animals immobilised for 7 days to keep a significantly greater absolute maximum isometric force and improves their performance (+21%, p=0.01) compared with animals treated with the vehicle, and this despite the absence of any effect of the compound BIO101 on the loss of mass.

[0112] It is observed that the animals treated with the vehicle have a specific maximum isometric contraction force (sP0; FIG. 5B) significantly less than that of the non-immobilised control animals (−13.2%, p<0.01). Remarkably, the specific maximum isometric force of the animals treated with the compound BIO101 is not significantly affected by 7 days of immobilisation: this is because the treatment with the compound BIO101 enables the animals immobilised for 7 days to keep a normal muscle function compared with the animals of the immobilised group, treated with the vehicle (+24.3%, p<0.001).

[0113] At the moment the immobilisation stops, at J14, the mice that receive the BIO101 treatment have lost only 22.4% (p<0.001) of the absolute maximum isometric force compared with the non-immobilised control mice (J0), as against 34% (p<0.001) for the mice that received the vehicle. Treatment with BIO101 limits the loss of absolute maximum isometric force (+17.5%, p<0.05) compared with mice treated with the vehicle (FIG. 6A).

[0114] Concerning the specific maximum isometric force, the mice that received the BIO101 treatment do not lose any force 14 days post immobilisation compared with the non-immobilised control mice (+5%, 2.94 g/mg versus 2.80 g/mg respectively, p=ns).

[0115] The mice treated with the vehicle for their part lose 11.3% of their specific force compared with the non-immobilised mice (p=0.06).

[0116] After 14 days of immobilisation, the treatment with BIO101 tends to limit the loss of specific maximum force (+18.4%, ns) compared with mice treated with the vehicle (FIG. 6B).

[0117] Because of the properties of phytoecdysones and derivatives thereof on the muscle function of mammals subjected to immobilisation, the use of phytoecdysones and derivatives thereof can therefore be proposed, for preserving muscle function, in particular with regard to muscle force, and thus slowing down the loss of muscle functions related to immobilisation.