A COMPOSITION HAVING A MUSCLE-RELAXANT AND ANTI-INFLAMMATORY ACTIVITY

Abstract

A pharmaceutical composition or dietary supplement having a muscle-relaxant and anti-inflammatory activity is described, which is effective in the treatment of muscle, bone, articular and tendon pathologies, comprising, as active ingredients, a combination of magnesium, at least one ketoboswellic acid and L-tryptophan.

Claims

1. A pharmaceutical composition or dietary supplement comprising, as the active ingredients, the combination of magnesium, L-tryptophan and at least one ketoboswellic acid, for use in a muscle-relaxant and anti-inflammatory treatment.

2. The pharmaceutical composition or dietary supplement according to claim 1, wherein said at least one ketoboswellic acid is 3-acetyl-11-keto-β-boswellic acid (AKBA).

3. The pharmaceutical composition or dietary supplement according to claim 1, for use in the treatment of muscle, bone, articular or tendon pathologies.

4. The pharmaceutical composition or dietary supplement according to claim 1, for use in the treatment of fibromyalgia, tension-type headache, poor-posture muscle pain, contracture, or for use in post-traumatic rehabilitation therapy.

5. The pharmaceutical composition or dietary supplement according to claim 1, comprising a Boswellia serrata dry extract as a source of 3-acetyl-11-keto-β-boswellic acid (AKBA), preferably a Boswellia serrata dry extract having a 3-acetyl-11-keto-β-boswellic acid (AKBA) titre of 30%.

6. The pharmaceutical composition or dietary supplement according to claim 1, wherein magnesium is in the form of magnesium bisglycinate.

7. The pharmaceutical composition or dietary supplement according to claim 1, in a dosage form comprising from 150 to 500 mg of magnesium, from 100 to 300 mg of Boswellia serrata extract and from 100 to 300 mg of L-tryptophan.

8. The pharmaceutical composition or dietary supplement according to claim 7, comprising about 225 mg of magnesium, about 175 mg of Boswellia serrata extract and about 150 mg of L-tryptophan.

9. The pharmaceutical composition or dietary supplement according to claim 1, which is in an oral dosage form.

10. The pharmaceutical composition or dietary supplement according to claim 1, for use in oral administration, wherein said use comprises the administration of from 150 to 1000 mg/day of magnesium, from 100 to 600 mg/day of Boswellia serrata extract and from 100 to 600 mg/day of L-tryptophan.

11. The composition according to claim 1, further comprising pharmaceutically acceptable excipients and/or binders and/or vehicles.

Description

[0018] The experimental section that follows describes studies that were performed relating to the biological effects of the composition of the invention. In the description of the studies performed, reference is made to the attached figures, in which:

[0019] FIG. 1 shows the results of the MTT test on THP-1 cells incubated with Milesax; the absorbance at 595 nm was measured in the various wells incubated for 16 hours with the indicated compounds at the indicated concentrations; each bar represents the average of at least 4 different wells (***p<0.001 relative to the vehicle Milesax);

[0020] FIG. 2 shows the results of the quantification of TNFα in the culture medium of differentiated THP-1 monocytes after pre-incubation with MILESAX and the various controls in the absence of an inflammatory stimulus (A) or following stimulation with LPS 1 μg/ml for 2 hours (B). (***=p<0.001;*=p<0.05);

[0021] FIG. 3 shows the results of the quantification of IL-1β in the culture medium of differentiated THP-1 monocytes after pre-incubation with MILESAX and the various controls in the absence of an inflammatory stimulus (A) or following stimulation with LPS 1 μg/ml for 2 hours (B). (***=p<0.001;*=p<0.05);

[0022] FIG. 4 shows the results of the quantification of IL-6 in the culture medium of differentiated THP-1 monocytes after pre-incubation with MILESAX and the various controls in the absence of an inflammatory stimulus (A) or following stimulation with LPS 1 μg/ml for 2 hours (B);

[0023] FIG. 5 shows the results of the quantification of the contractions per minute (A) and of the average displacement of the myotube calculated as the distance traveled during a single contraction taken at an easily identifiable point of the cell (B);

[0024] FIG. 6 shows the results of another experiment for quantifying the contractions per minute (A) and the average displacement of the myotube calculated as the distance traveled during a single contraction taken at an easily identifiable point of the cell (B).

[0025] A particularly preferred formulation of the composition of the invention, used in the experimental studies, is also given hereinbelow. In addition to the indicated active ingredients, the formulation comprises suitable excipients and optionally flavourings and colorants.

FORMULATION EXAMPLE—4.5 g SACHET

[0026]

TABLE-US-00001 Magnesium 225 mg Boswellia serrata extr. 175 mg of which AKBA 52.5 mg L-Tryptophan 150 mg

[0027] The recommended dose is one or two sachets per day.

EXPERIMENTAL SECTION

Rationale

[0028] The composition described above in the formulation example, referred to hereinabove as MILESAX, was used in the experimental study illustrated in the present description.

[0029] Extracts from the gum resin of Boswellia serrata and certain components thereof such as boswellic acids (BAs) may influence the immune system in various ways. For example, BAs may interact with the production and release of cytokines. It has been reported that BAs inhibit activation of the transcription factor NF-kB giving rise to a reduction in TNFα and various other cytokines, such as the interleukins IL-1β, IL-2, IL-4, IL-6 (Ammon H. P., 2010, Phytomedicine, 17(11): 862-7). In a similar manner to BAs, an endogenous tryptophan metabolite (5-methoxytryptophan) may also have anti-inflammatory action, regulating the expression of cyclooxygenase-2 (COX-2), the enzyme responsible for the synthesis of prostaglandins following inflammatory stimuli (Cheng H. H. et al., 2012, PNAS 109(33): 13231-6). It is also known that magnesium may have muscle-relaxant effects mediated by inhibition of the type-L calcium channels in smooth muscle cells of rabbit arteries (Sharma N. et al., 2012, Neurol. Res. 34(3): 291-6), and as such it is capable of reducing spontaneous contractions and contractions induced by oxytocin in muscle cells derived from the endometrium (Tica V. I. et al., 2007, Gynecol. Endocrinol. 23(7): 368-72). Magnesium is also known as an activator of the calcium-activated potassium channels, which are essential for modulating muscle contraction and neuronal activity (Shi J. et al., 2002, Nature 418: 876-880) and variations in magnesium concentration may modulate the contractility of cardiac muscle (Puchert E. et al., 2003, Pflugers Arch. 447(2): 135-41).

OBJECT OF THE STUDY

[0030] In the present experimental study, the anti-inflammatory activity of MILESAX in human monocyte cell cultures (THP-1 cells) following stimulation with LPS (lipopolysaccharide, one of the components of the outer membrane of Gram-negative bacteria), was analysed. The capacity of MILESAX to inhibit transduction of the signal which, from the extracellular stimulus (LPS), leads to the production of the cytokines TNF-α, IL-1β and IL-6 was evaluated quantitatively by means of an Elisa test (Test 1).

[0031] The muscle-relaxant activity of MILESAX in primary cell cultures of human skeletal muscle was also analysed, as was the capacity of the compound to modulate the contractile activity of differentiated myotubes in culture following stimulation with carbacol, a non-hydrolysable analogue of acetylcholine (neurotransmitter of the neuromuscular synapse).

Materials and Methods

Milesax

[0032] Since the individual components of MILESAX do not have the same solubility characteristics, they were each dissolved in the appropriate solvent and then mixed in the identical proportions of MILESAX to reconstitute the whole compound, as illustrated in Table 1 below.

TABLE-US-00002 TABLE 1 [final] Component in mM solvent Magnesium 1.0 Medium B. serrata 0.3 DMSO L-tryptophan 0.6 H.sub.2O

[0033] The concentration of magnesium 1 mM was taken as reference (Suzuki-Kakisaka et al., American Journal of Reproductive Immunology 70 (2013) 213-220).

[0034] The positive controls with which were compared the effects produced by MILESAX in the various tests were: diclofenac and ibuprofen for the anti-inflammatory effect (Test 1), eperisone hydrochloride and thiocolchicoside for the muscle-relaxant effect (Test 2).

Cells

[0035] Human THP-1 cells derived from acute monocytic leukaemia (ATCC, cat. N. TIB-202) were cultured in RPMI-1640 medium (LifeTechnologies, cat. N. 21870-076) with the addition of sodium pyruvate 1 mM, HEPES 10 mM, L-glutamine 2 mM, 2-mercaptoethanol 0.05 mM, and foetal bovine serum to a final concentration of 10%. The cells were maintained in an incubator at 37° C. and 5% CO.sub.2 and seeded every 3-4 days at a density of about 5×10.sup.5 cells/ml.

[0036] For all the following tests, the cells were seeded in the evening in 96-well multi-well plates and incubated with various concentrations of MILESAX, of its vehicle, of the positive control(s) in whole medium.

[0037] After about 16 hours of incubation, the cells were differentiated in serum-free medium for 2 hours in the presence or absence of LPS (1 μg/ml).

[0038] The human skeletal muscle cells (Clonetics™ Skeletal Muscle Myoblast Cell Systems, Lonza) were cultured in Skeletal Muscle Growth Media-2 (CC-3244, Lonza) containing hEGF (human Epidermal Growth Factor), dexamethasone, L-glutamine, foetal bovine serum and gentamicin/amphotericin B. The cells were maintained in an incubator at 37° C. and 5% CO.sub.2 and seeded every 3-4 days at a density of about 3500 cells/cm.sup.2, the medium being exchanged with fresh medium daily. The myoblasts were differentiated into myotubes in differentiation medium (DMEM: F12, with the addition of 2% of horse serum). The differentiated myotubes were used for the experiments from the fourth to the seventh day of maintenance in differentiation medium.

MTT Test (Test 0)

[0039] To check that the incubation with MILESAX at the concentrations and for the times chosen was not toxic to the cells, one or more MTT tests (Test 0) were performed. This colorimetric test is based on the transformation of the tetrazolium salt MTT (yellow) into formazan (violet), by the succinate-tetrazolium reductase system, which belongs to the mitochondrial respiratory chain and is active only in metabolically active cells. Briefly, the cells grown in a 96-well multi-well plate were incubated with MTT solution for 4 hours.

[0040] An insoluble colorant forms during this period, which, after dissolving by adding dissolution solution to the samples (10% SDS in 10 mM HCl) and after incubating overnight in an incubator, may be quantified by reading the absorbance of the samples at 595 nm (using 750 nm as reference wavelength). The absorbance measured correlates directly with the number of live cells.

ELISA Test (Test 1)

[0041] The anti-inflammatory effect of MILESAX was studied via an ELISA test (Enzyme-Linked Immunosorbent Assay, Biolegend, Inc.), by quantifying the production of pro-inflammatory cytokines in the culture medium following treatment with MILESAX and with various controls.

[0042] In the “sandwich” ELISA test, a 96-well plate is covered with a monoclonal antibody specific for a given cytokine. The standards and samples are added to the wells and the cytokine of interest bonds the uptake antibody immobilized to the bottom of the well. Next, an anti-cytokine biotinylated antibody is added to the wells so that an antibody-antigen-antibody “sandwich” is formed. Horseradish peroxidase combined with streptavidin is then added, followed by a solution of tetramethylbenzidene (TMB), which, by reacting with the peroxide, produces a blue-coloured compound, the intensity of which colour is proportional to the amount of cytokine present. The addition of a sulfuric acid solution changes the colour of the solution from blue to yellow, blocking the development of the colour and allowing accurate reading of the absorbance of the samples at 450 nm.

[0043] The cells incubated with the various overnight treatments were then differentiated for 2 hours in serum-free medium and simultaneously stimulated with LPS (lipopolysaccharide, one of the components of the outer membrane of Gram-negative bacteria). At the end of the treatment, the supernatant was collected and stored at −80° C. until the time of use for the test. The cells were used to measure the total protein content in the various samples. The concentrations of cytokines released into the culture medium were then expressed as pg of cytokine/mg of total protein of the sample. In this way, any differences in cytokine concentration in the medium due to a different amount of cells in the samples were eliminated.

Evaluation of the Muscle-Relaxant Effect (Test 2)

[0044] For the evaluation of the muscle-relaxant effect of MILESAX, human myoblasts were seeded onto microscope slides and, once they had reached at least about 70% confluence, they were maintained in differentiation medium for 4-7 days before being used for the experiments. The contractile activity of the myotubes was recorded with a TCS SP5 confocal microscope (Leica Microsystems) in transmitted-light mode with the 40× objective lens (HCX PL APO, 1.25 NA, Leica Microsystems) using the incubator associated with the microscope to maintain the cells under a controlled atmosphere (37° C., 5% CO.sub.2).

[0045] The myotubes were stimulated with 3 mM of carbacol, dissolved in differentiation medium, for at least 20 minutes before starting the recordings and maintained in differentiation medium containing carbacol and the various treatments throughout the experiments.

Statistical Analysis

[0046] The quantitative data relating to the various tests were expressed as the mean±standard error. The one-way ANOVA statistical test (comparison between more than two groups) or the unpaired T test (comparison between two groups) were used to reveal statistically significant differences between the various samples.

Results

Identification Doses and Treatment Times (Test 0)

[0047] The THP-1 cells were incubated overnight with successive dilutions of MILESAX, starting from the composition having as reference a magnesium concentration of 1 mM. To exclude a possible toxic effect attributable to the chosen incubation protocol, the cell vitality was quantified by means of an MTT test. The data obtained in two different experimental days are collated in FIG. 1. Since the first tested concentrations of MILESAX (0.5, 0.75 and 1 mM) showed high cell mortality, the following tests were performed with MILESAX 50, 75 and 100 μM. At these concentrations of compound, the measured absorbance is not statistically different from that of the control (cells incubated with the MILESAX vehicle referred to the highest concentration).

[0048] In the same manner, incubation with the positive controls diclofenac and ibuprofen at a concentration of 100 μM (Mouuthys-Mickalad A. et al., BBRC, 2004, vol. 325, pages 1122-30) showed no toxic effect on the cells. The concentrations of the individual components in the three dilutions of MILESAX used in the following tests are given below, in Table 2:

TABLE-US-00003 TABLE 2 Concentration of the individual components of MILESAX in the three dilutions used for tests 1 and 2. [final] [final] [final] Component in mM in mM in mM Magnesium (from magnesium bisglycinate) 0.1 0.075 0.05 Boswellia serrata 0.03 0.0225 0.015 L-Tryptophan 0.06 0.045 0.03

Anti-Inflammatory Activity in a Human Monocyte Cell Line (Test 1)

[0049] The anti-inflammatory activity of MILESAX was studied by quantifying the inhibition of production of pro-inflammatory cytokines by means of an ELISA test (Test 1).

[0050] Release into the culture medium of the pro-inflammatory cytokines TNFα, IL-1β and IL-6 from the differentiated THP-1 monocytes was evaluated both in the absence and in the presence of stimulation with LPS for 2 hours. The data obtained for TNFα are collated in FIG. 2.

[0051] In the absence of inflammatory stimulus, the production of TNFα was very low, but, as expected, was appreciably amplified by stimulation with LPS.

[0052] Incubation for 16 hours with MILESAX at all the tested concentrations (50, 75 and 100 μM) led to a statistically significant reduction in the release of TNFα relative to the control condition (incubation with the MILESAX vehicle, white bar). The release of TNFα into the culture medium following incubation with the three concentrations of MILESAX was significantly smaller compared with the positive controls diclofenac and ibuprofen (p<0.001, one-way ANOVA test). While incubation with diclofenac 100 μM significantly reduced the release of TNFα relative to incubation solely with diclofenac vehicle (p<0.05, T test), incubation with ibuprofen 100 μM did not lead to any reduction in the release of TNFα relative to the control.

[0053] The data relating to the quantification of IL-1β are collated in FIG. 3. As for TNFα, the quantification of IL-1β also showed a significant increase in the release following inflammatory stimulus with LPS, although smaller than the increase measured for TNFα. In this case also, the reduction in IL-1β release was statistically significant relative to the control at all the MILESAX tested concentrations and similar to or better than that for diclofenac, which was, nevertheless, effective in reducing the release of IL-1β. Ibuprofen, in contrast, had no effect on the release of IL-1β.

[0054] The data relating to the quantification of IL-6 are collated in FIG. 4. The release of IL-6 was not significantly stimulated by stimulation with LPS, probably requiring longer incubation times. Although a tendency of the compounds to reduce the production of cytokines by the monocytes was observed, no effect was statistically significant, except for the comparison between the three MILESAX concentrations and the cells alone (p<0.001 for MILESAX 50 μM and p<0.01 for MILESAX 75 and 100 μM).

Evaluation of the Muscle-Relaxant Effect (Test 2)

[0055] Human muscle myoblasts in culture are capable of differentiating into multinuclear myotubes that are capable of contracting following electrical stimuli or exposure to acetylcholine receptor agonists.

[0056] Not all the myotubes in the culture were capable of contracting, and similarly it was never possible to observe spontaneous contractions only in differentiation medium. For this reason, the differentiated myotubes were stimulated with carbacol 3 mM in differentiation medium. The contractile activity of the identified active cell(s) was recorded with a confocal microscope in transmitted light, with an image acquired every 151 ms. After the first 5 minutes of controlling the activity of the myotube in differentiation medium, the same cell was exposed to the various treatments, MILESAX or thiocolchicoside (Muscoril) and washing (wash, exposure once again to differentiation medium). Carbacol was present throughout the recordings.

[0057] Modulation of the contractile activity of the myotubes was quantified by analysing two parameters: the number of contractions per minute and the mean displacement of the myotube during the contractile activity (Manabe Y. et al., 2012, PLOS ONE 7(12): 1-10).

[0058] The experiment performed revealed that there was a clear tendency both in the positive control Muscoril and in MILESAX to reduce the frequency of contractions per unit time and that the frequency of contractions was at least partly restored during washing (wash, exposure once again to differentiation medium, in the presence of carbacol, but in the absence of the treatments, FIG. 5A).

[0059] The mean displacement of the myotube during contraction is similar to the data noted in the literature (Manabe Y. et al., 2012, PLOS ONE 7(12): 1-10) and does not undergo variations during the treatments (FIG. 5B).

[0060] The quantification of another experiment, in which the cell was exposed to MILESAX, is illustrated in FIG. 6. In this case, the effect of MILESAX on reducing the number of contractions per minute was statistically significant relative to the control. The myotube partly recovers the contraction frequency observed in the control after removal of MILESAX and exposure once again to differentiation medium.

[0061] The myotubes which did not at least partly recover contractile activity after exposure to the treatments and during washing were not considered.

[0062] Since the analysis of the contractile activity in the “live” experiments illustrated herein required long microscope acquisition times, to ensure the reliability of the reported data, it was not possible to compare the effects of MILESAX with the two positive controls suggested by the client, but only with one of the two (thiocolchicoside—Muscoril).

CONCLUSIONS

[0063] In conclusion, MILESAX demonstrated both anti-inflammatory properties in the THP-1 human monocyte cell model and muscle-relaxant properties in the human primary myotube cell model used for this study.