THERAPY FOR PROMOTING CELL GROWTH

Abstract

There is disclosed the use of a composition for promoting neuronal growth of neurons in tissues of the central or peripheral nervous system. There is also disclosed a method for inducing proliferation or differentiation of neuronal cells.

Claims

1.-73. (canceled)

74. A composition comprising the following four herbal components: i) Radix Astragali; ii) Radix Polygalae; iii) Rhizoma Chuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and iv are present in the composition in a ratio of about 5:1:1:1 by weight, respectively.

75. A composition consisting essentially of the following four herbal components: i) Radix Astragali; ii) Radix Polygalae; iii) Rhizoma Chuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and iv are present in the composition in a ratio of about 5:1:1:1 by weight, respectively.

76. An extraction product of a composition comprising the following four herbal components: i) Radix Astragali; ii) Radix Polygalae; iii) Rhizoma Chuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and iv are present in the composition in a ratio of about 5:1:1:1 by weight, respectively.

77. An extraction product of a composition consisting essentially of the following four herbal components: i) Radix Astragali; ii) Radix Polygalae; iii) Rhizoma Chuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and iv are present in the composition in a ratio of about 5:1:1:1 by weight, respectively.

78. The composition according to claim 74, wherein said composition is a pharmaceutical composition comprising a pharmaceutically acceptable additive, carrier or diluent.

79. The composition according to claim 75, wherein said composition is a pharmaceutical composition comprising a pharmaceutically acceptable additive, carrier or diluent.

80. The product according to claim 76, wherein said product is a pharmaceutical product comprising a pharmaceutically acceptable additive, carrier or diluent.

81. The product according to claim 77, wherein said product is a pharmaceutical product comprising a pharmaceutically acceptable additive, carrier or diluent.

82. The product according to claim 80, wherein said pharmaceutical product is administered orally.

83. The product according to claim 81, wherein said pharmaceutical product is administered orally.

84. The product according to claim 80, wherein said pharmaceutical product is administered orally as one or more tablets or capsules.

85. The product according to claim 81, wherein said pharmaceutical product is administered orally as one or more tablets or capsules.

86. The product according to claim 80, wherein said pharmaceutical product is used in combination with one or more Western medicines.

87. The product according to claim 81, wherein said pharmaceutical product is used in combination with one or more Western medicines.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 shows bar charts of the dose-effect of NeuroAid™ (MLC 601) treatment on cell viability on day 8, day 10 and day 14. Three concentrations of NeuroAid™ (MLC 601) (0.1, 0.5, 1.0 μg/ml) were compared to the control.

[0057] FIG. 2 shows bar charts of the effect of NeuroAid™ (MLC 601) treatment on LDH release on day 10 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) was compared to the control.

[0058] FIG. 3 shows confocal images of the effect of NeuroAid™ (MLC 601) treatment on neuronal DCX expression on day 3 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) was compared to the control.

[0059] FIG. 4 shows bar charts of the effect of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments on cell viability on day 8, day 10, day 12 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0 μg/ml NeuroAid II (MLC 901) were compared to the control.

[0060] FIG. 5 shows a line graph of the effect of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments on LDH release on day 8, day 10, day 12 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0 μg/ml NeuroAid II (MLC 901) were compared to the control.

[0061] FIG. 6 shows confocal images of the effect of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments on DCX expression on day 3 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0 μg/ml NeuroAid II (MLC 901) were compared to the control.

[0062] FIG. 7 shows schematic diagrams of the experimental protocols for NeuroAid II (MLC 901) treatments against stroke. FIG. 7A shows the experimental protocol for Example 4, which investigates the effects of NeuroAid II (MLC 901) treatment; FIG. 7B shows the experimental protocol for Example 5, which investigates the effects of an acute NeuroAid II (MLC 901) treatment. Both studies assessed the rate of survival and also quantified the infarct volume at 24 h post-ischemia.

[0063] FIG. 8 shows a bar graph of the survival rate after NeuroAid II (MLC 901) pre-treatment at 24 h post-ischemia.

[0064] FIG. 9 shows a bar graph of the survival rate (A) and infarct volume (B) after NeuroAid II (MLC 901) post-treatment at 24 h post-ischemia.

[0065] FIG. 10 shows bar charts on the comparative effects of NeuroAid II (MLC 901), Rhizoma chuanxiong Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis treatments (1 μg/ml) on cortical neurons in culture estimated at Day 12 of treatment. FIG. 10A shows the effect of the respective treatments on cell viability and FIG. 10B shows the effect of the respective treatments on LDH release.

[0066] FIG. 11 shows bar charts on the comparative effects of a post-treatment with NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis against ischemic brain injury in vivo. FIG. 11A and FIG. 11B shows the survival rate (A) and infarct volume (B) respectively.

[0067] FIG. 12 shows bar charts on the comparative effects of a pre-treatment with NeuroAid II (MLC 901), Rhizoma chuanxiong and Radix angelicae sinensis against ischemic brain injury in vivo. FIG. 12A and FIG. 12B shows the survival rate (A) and infarct volume (B) respectively.

[0068] FIG. 13 shows representative epifluorecence microscopy photographs of the comparative effects between NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis treatment (1 μg/ml) on in vitro DCX immunoexpression in cultured cortical cells at Day 12 of treatment.

[0069] FIG. 14 shows bar charts of DCX signal intensity in immunostained neurons observed in epifluorecence microscopy.

[0070] FIG. 15 shows bar charts of the neurite outgrowth obtained by measuring on epifluorecence microscopy, the total length of neurite WO in function of treatments.

EXAMPLES

[0071] Non-limiting examples of the invention will be further described in greater detail by reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Materials

Medium for Cortical Sampling:

[0072] HBSS/I

[0073] NaCl 8 g/I

[0074] KCl 0.26 g/I

[0075] MgSO.sub.4 0.2 g/I

[0076] CaCl.sub.2 0.264 g/I

[0077] NaH.sub.2CO.sub.3 2.24 g/I

[0078] NaH.sub.2PO.sub.4 0.15 g/I in H.sub.2O [0079] HBSS+: add 6 g glucose

Cell Culture Medium

[0080] Neurobasal (21103-049; Invitrogen, San Diego, Calif.)

[0081] qB27 (n° 17504; Invitrogen, San Diego, Calif.)

[0082] Uridine (U3003; Sigma, St. Louis, Mo., USA)

[0083] Fluoro-deoxy-Uridine (46875; Fluka-Chemika-Biochemika, Buchs, Switzerland)

[0084] Glutamax (35050; Gibco-BRL Life Technologies. GmbH, Karlsrahe, Germany)

[0085] Antibiotics (Penicillin-streptomycin) (Gibco-BRL Life Technologies. GmbH, Karlsrahe, Germany)

Animals

[0086] All experiments were performed according to policies on the care and use of laboratory animals of European Community legislation. The local Ethics Committee approved the experiments (protocol numbers NCA/2006/10-1 and NCA/2006/10-2). Adult male C57/B16 mice, weighing 22 to 26 g were used in this study. The animals housed under controlled laboratory conditions with a 12-hour dark-light cycle, a temperature of 21±2° C., and a humidity of 60 to 70% for at least one week prior to drug treatment or surgery. The mice had free access to standard rodent diet and tap water.

NeuroAid II (MLC 901) Drinking Solution

[0087] One capsule of NeuroAid II (MLC 901) was dissolved in 66 ml water under stirring with an agitator for one hour at 37° C. The solution is then filtered with a 0.45 μm filter.

NeuroAid II (MLC 901) for Intraperitoneal Injections

[0088] 30 mg from a NeuroAid II (MLC 901) capsule was diluted in 3 ml saline solution corresponding to a concentration of 10 mg/ml (Stock solution) at 37° C. during 60 min. The concentration used in the experiment is 1 μg/ml (injected volume: 500 μl). To obtain the dose of 1 μg/ml, the stock solution is diluted by 100. After dilution, the mixture is vortexed to obtain a good homogenization and filtered on a 0.45 μm filter.

Methods

A. Cortical Cell Culture

[0089] Time-pregnant (E14) C57B16/J mice were anesthetized with isopentane followed by cervical dislocation. Fetuses were removed and placed in cold (Hanks Balanced Salt Solution) HBSS+solution. Cerebral cortices were dissected in cold HBSS+solution and the meninges were removed. The cortical samples were cut in small pieces and were gently triturated with a fire-polished glass Pasteur pipette in 8 nil HBSS+solution. The mix was filtered (40 μM filter) and centrifuged at 800 rpm for 8 min. The supernatant was removed and the pellet was dissolved in 2 ml culture medium. Cells were plated on Polylysine-coated 12 well (24 mm diameter; Sigma-Aldrich Chimie, St. Quentin Fallavier, France)) plates with glass coverslips (12 mm diameter; CML, Nemours, France) at a density of 1×10.sup.6 cells/well. Cultures were maintained at 37° C. in a humidified 5% CO.sub.2 atmosphere incubator in Neurobasal supplemented with B27, Glutamax, antibiotics were used for experiments after 16 days. Glial growth was suppressed by addition of 5-Fluoro-2-deoxyuridine (2 μM) and Uridine (2 μM) during the second day of culture.

B. Neuroaid™ (MLC 601) Treatment

[0090] 30 mg from a NeuroAid™ (MLC 601) capsule was diluted in 3 ml Neurobasal medium corresponding to a concentration of 10 mg/ml (Stock solution) at 37° C. for a duration of 60 minutes. The concentrations tested in the experiments were: 1 μg/ml or 10 μg/ml of culture medium. 1 ml of NeuroAid™ (MLC 601) solution was put in each 24 mm well of plate.

[0091] To obtain the dose of 1 μg/ml, the stock solution was diluted by 100 times: 0.1 mg/ml (10 μl per 24 mm well/ml, corresponding to 1 μg/well).

[0092] To obtain the dose of 10 μg/ml, the stock solution was diluted by 10 times: 1 mg/ml (10 μl per 24 mm well/ml, corresponding to 10 μg/well).

[0093] After dilution, the mixture was vortexed to obtain a good homogenization and filtered on a 0.45 μm filter.

[0094] Cells were treated each day with NeuroAid™ (MLC 601), NeuroAid II (MLC 901) or Neurobasal medium from Day 1 of culture.

C. Lactate Dehydrogenase (LDH) Measurements

[0095] Neuronal injury was quantitatively assessed by the measurement of lactic dehydrogenase (LDH) release from cultured neurons incubated in cell culture medium. At Day 1, 5, 8, 10, 12, 14 and 16, 100 μl of the cell culture medium was transferred from culture wells to 96-well plates and mixed with 100 μl of reaction solution according to LDH assay kit (Roche Diagnostic: Cytotoxicity Detection kit: ref 1644793, Indianapolis, USA). Optical density (OD) was measured 30 minutes later at 492 nm utilizing a Labsystem Multiscan microplate reader (Labsystem Multiscan RC, Finland). Background absorbance at 620 is subtracted. LDH activity is expressed as activity present in the 25 μl medium volume. Results are expressed as OD×10.sup.−3.

D. Cell Viability

[0096] At Day 1, 5, 8, 10, 12, 14 and 16, the totality of cell culture medium was removed and replaced by 500 μl of Neurobasal medium+Cell Titer 96 Aqueous One Solution kit: Cell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay Kit. Neuronal viability was determined using the Cell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay (Promega, Madison, USA). According to the Proliferation Assay Kit protocol, cells are incubated for 4 hours at 37° C. in the humidified 5% CO.sub.2 atmosphere incubator. The reaction was stopped with 2% Sodium Dodecyl Sulfate (SDS). Optical density was measured 4 hours later at 490 nm utilizing a Labsystem Multiscan microplate reader. Background absorbance at 620 was subtracted. Results were expressed as OD×10.sup.−3 representating the number of viable cells.

[0097] Statistic analysis of cell viability and LDH results are assessed using one factor (analysis of variance) ANOVA test followed by post-hoc test (P<0.05).

E. Double Cortin (DCX) Immunohistochemistry of Cortical Cell on Coverslips

[0098] Cortical cell are fixed onto coverslips with 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized in 0.3% polyoxyethylensorbitan monolaurate (Tween 20, Sigma) for 10 minutes and blocked with 2.5% donkey serum in PBS for 2 hours at room temperature. Coverslips were incubated with a goat anti-doublecortin (DCX) antibody (1:200, SC-8066, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) in 2% donkey serum/PBS overnight. After 3 washes in PBS, coverslips were incubated in anti-goat Alexa 488-coupled antibodies (FluoProbes, Interchim, Montluçon, France) in 2% donkey serum for 2 hours, and washed three times in PBS for 5 min each. The coverslips are then incubated in Hoechst solution (3 μl in 10 ml; Sigma-Aldricht Chimie, Saint Quentin Fallavier, France) for 10 minutes to label cell nuclei. After 2 washes in PBS and 1 wash in water, the coverslips are dried and mounted on glass slides with Fluoroprep (75521; BioMérieux, Marcy l′Etoile, France). Cells were observed using epifluorescence and confocal microscopy. Confocal microscopy observations are performed using a Laser Scanning Confocal Microscope (TCS SP, Leica Microsystems Heidelberg GmbH, based in Mannheim, Germany) equipped with a DMIRBE inverted microscope and an argon-krypton laser (laser excitation 488 nm, acquisition 500-600 nm every 10 nm). Signal specificity was assessed in negative control coverslips by omitting primary antibody directed against the DCX protein. Images were acquired as single transcellular optical sections and averaged over at least four scans per frame. Confocal images of DCX-Alexa-488 antibody labeling were then obtained after spectral correction of the autofluorescence background.

F. Physiological Parameters Used when Investigating the Effects of a NeuroAid II (MLC 901) in C57BI/6 Mice

[0099] General anesthesia was induced with 3% isoflurane and maintained with 1% isoflurane by means of an open facemask for each mouse. Mice were allowed to breathe spontaneously. A subset of animals (n=5 per group) were monitored for physiological parameters including mean arterial blood pressure (MABP), rectal temperature, arterial blood gases and pH before, during and after ischemia. The right femoral artery was catheterized with PE-10 polyethylene tubing and connected to a blood pressure transducer (Harvard Apparatus, Massachusetts, USA) for continuous monitoring of MABP (mm Hg). A heparinized blood sample (75 1.11) was then obtained from the catheterized femoral artery and blood pO.sub.2, pCO.sub.2 and pH were measured using an Acid-Base Laboratory system (ABL 555, Radiometer). Core temperature was monitored continuously with a thermometer (3-mm probe diameter; Harvard Apparatus, Massachusetts, USA), inserted into the rectum and maintained at physiological temperatures using a thermostatically controlled heating blanket (Harvard Apparatus, Massachusetts, USA). Core temperature was maintained at physiological values by a combination of the homeothermic blanket control.

G. Induction of Transient Focal Cerebral Ischemia in C57131/6 Mice

[0100] Focal ischemia was induced by occlusion of the left middle cerebral artery (MCA) using an intraluminal filament technique (Heurteaux et al, 2006) After a midline neck incision was made, the left common and external carotid arteries were isolated and ligated with a silk 4-0 silk suture (Ethicon). A temporary yasargil aneurysm clip (BMH31, Aesculap, Tuttlingen, Germany) was temporarily placed on the internal carotid artery. A 6-0 coated filament (Doccol, Redlands, Calif., USA), blunted at tip with an open flame, was introduced through a small incision into the common carotid artery and 13 mm distal to the carotid bifurcation for occlusion of the origin of the MCA. Animals were kept at 37° C. for one hour, after which time the thread was carefully withdrawn to allow reperfusion of the MCA territory. To control the MCAO severity regional CBF (rCBF) was determined by laser-Doppler flowmetry (Perimed) using a flexible 0.5-mm fiber optic extension to the masterprobe fixed on the intact skull over the ischemic cortex (2 mm posterior and 6 mm lateral from the bregma). Sham-operation was performed inserting the thread into the common carotid artery without advancing it to occlude the MCA. The animals were allowed to regain full consciousness on a heating pad before returning to the cage.

H. Determination of Infarct Volume

[0101] To assess the infarct volume in the acute NeuroAid II (MLC 901) post-treatment study (Example 5), mice were sacrificed at 24 hours after reperfusion. Their brains were removed and sectioned into six 1 mm-thick coronal slices using a tissue chopper (Phymep, France). Coronal brain slices were immediately immersed into 2% 2, 3, 5-Triphenyltetrazolium chloride (TTC, Sigma, France) for 20 min at room temperature in the dark followed by fixation in a 4% paraformaldehyde solution overnight prior to analysis as described previously (Ding-Zhou et al., 2002). The striatal and cortical areas of infarction, outlined in light were measured on each section using a computer image analysis system and corrected for brain edema according to Golanov and Reis (Golanov and Reis, 1995). Infarct volume, expressed in mm.sup.3 was calculated by a linear integration of the corrected lesions areas.

[0102] In addition cresyl violet, a dye that stains the Nissl bodies in the stellate somas of viable neurons was used to confirm the evolution of infarct volume in ischemic mice. Coronal frozen sections of brain (10 μm-thick) were added to a solution of 1% cresyl violet in 0.25% acetic acid for 3 min, rinsed, dehydrated and mounted with Entellan. Sections were analyzed under light microscopy.

Example 1

Dose-Effect of NeuroAid™ (MLC 601) on Cell Viability and LDH Release

[0103] Cortical cells, as prepared by the methods disclosed above, were exposed to four concentrations of NeuroAid™ (MLC 601): 0.1, 0.5, 1.0, 10 μg/ml from Day 1 until Day 14 of culture. Cell viability was studied at Day 8, 10 and 14 by the methods disclosed above and the results are shown in FIG. 1.

[0104] The effect of NeuroAid™ (MLC 601) treatment against neurodegeneration of cortical cells over time in culture was also analyzed by LDH release. Increasing cell suffering that leads to cell death is associated with increased LDH release. LDH release was measured on day 10 and day 14 by the methods disclosed above and the results are shown in FIG. 2.

Results

[0105] FIG. 1 shows that at Day 14, a NeuroAid™ (MLC 601) treatment at a concentration of 1 μg/ml induced a significant increase (35%) in neuronal survival as compared to control (**P<0.01). Until Day 10 there was no significant difference in the percentage of cell viability on cells treated with NeuroAid™ (MLC 601) concentrations of 0.1-0.5-1.0 μg/ml as compared to control (P>0.05) (n=10 wells per experimental group).

[0106] FIG. 2 shows the ratio of LDH release/cell viability at Day 10 and 14 at the concentrations of 1 μg/ml NeuroAid™ (MLC 601). Compared to control, NeuroAid™ (MLC 601) treatment significantly reduced the LDH release after 10 and 14 days of culture (P<0.01) (n=10 wells per experimental group).

[0107] Based on the experimental data in Example 1, it can be demonstrated that treatment with NeuroAid™ (MLC 601) in cortical cells in culture demonstrates an increase of cell viability and a reduction of LDH release, which is a marker of the cell suffering. Example 1 also demonstrates that NeuroAid™ (MLC 601) may be used for diminishing the effect of stroke or neurodegeneration in predisposed subjects or subjects at risk of stroke or neurodegeneration, for treating patients with diseased or injured tissues of the central or peripheral nervous systems, or for use as a cell culture reagent.

Example 2

Effect of NeuroAid™ (MLC 601) on Neuronal Proliferation/Neurogenesis

[0108] DCX is a highly hydrophilic microtubule-associated protein that was expressed specifically in all migrating precursors of the developing CNS and in areas of continuous neurogenesis in adult brain. Neuronal proliferation was analyzed from DCX expression in cortical cells in culture by the methods disclosed above. Cortical cells, as prepared by the methods disclosed above, were treated with 1 μg/ml NeuroAid™ (MLC 601) from Day 1 until Day 14 of culture. The cells were then prepared and observed using epifluorescence and confocal microscopy as described in the methods above to determine DCX expression. The results are shown in FIG. 3.

Results

[0109] In FIG. 3, staining with an antibody against DCX shows that at Day 3, there was no difference in DCX expression between the control and cortical cells treated with 1 μg/ml NeuroAid™ (MLC 601). However, at Day 14, DCX immunoreactivity disappeared in the control, while there was an increase of DCX expression induced by NeuroAid™ (MLC 601) treatment, highlighting the development of an important axonal and dendritic network.

[0110] Because of its association with neurogenic processes, the DCX protein is currently used as a marker for neurogenesis. DCX is a microtubule-associated protein whose expression is associated with all migrating neuronal precursors in fetal and adult brain. DCX appears to be important for the normal developmental migration of cortical neurons, because mutations in DCX in humans lead to syndromes characterized by migrational arrest of these neurons and which is manifested clinically by subcortical laminar heterotopias, mental retardation and seizures. DCX is also expressed in some mature neurons in the adult brain, where it is involved in axonal outgrowth and synaptogenesis.

[0111] Thus, based on the experimental data of Example 2, it can be demonstrated that treatment with NeuroAid™ (MLC 601) results in an increase in DCX expression in cortical cells, suggesting strongly that treatment with NeuroAid™ (MLC 601) enhances neuroproliferation, neurogenesis, and neurorepair necessary to restore neurological function (such as motor and cognitive). Example 2 also demonstrates that NeuroAid™ (MLC 601) may be used for promoting neuronal outgrowth and proliferation of neurons in tissues of the central or peripheral nervous systems, for promoting cell growth, and for a method for inducing cell survival, growth, proliferation or differentiation of cells using NeuroAid™ (MLC 601).

Example 3

Effect of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) Treatments on Cell Viability, LDH Release and Neuronal Proliferation

[0112] Cortical cells were exposed to a concentration of 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC 901) from Day 1 until Day 14 of culture. This concentration of 1 μg/ml corresponds to the best results obtained on cell viability and LDH release as described above. Cell viability was studied at Day 8, 10, 12 and 14. Neuronal proliferation in the course of time was analyzed by observing DCX expression in cortical cells in culture treated with 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC 901).

Results

[0113] FIG. 4 shows that at Day 8, a significant increase in neuronal viability as compared to respective control (**P<0.01; ***P<0.001) was observed in cortical cells treated with 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC 901). However, the highest efficiency of both treatment was observed at Day 14 with 45% increase of cell survival (***P<0.001). There was no significant difference of efficiency between NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) at the different stages of culture (n=10 wells per experimental group).

[0114] FIG. 5 shows the comparison of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments on the ratio of LDH release/cell viability at Day 8, 10, 12 and 14 at the concentrations of 1 μg/ml. Compared to the control, both treatments significantly reduced LDH release after 12 and 14 days of culture (*P<0.05 and ***P<0.001) (n=10 wells per experimental group). There was no significant difference of efficiency on LDH release between NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments.

[0115] FIG. 6 shows that, compared to the control, cortical cells treated with 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC 901) developed a much denser axonal and dendritic network from Day 3 in culture. At Day 14, DCX immunoreactivity disappeared in Control and the labeling of the DCX protein became different between cortical cells treated with NeuroAid™ (MLC 601) and cortical cells treated with NeuroAid II (MLC 901). The DCX protein was always expressed in processes of cortical cells treated by NeuroAid™ (MLC 601) or NeuroAid II (MLC 901). However, it appeared that in cortical cells treated with NeuroAid II (MLC 901), there was also a strong increase of DCX immunoreactivity in the cytoplasm of these cells.

[0116] The results obtained with both NeuroAid™ (MLC 601) treatments (NeuroAid™ (MLC 601) and NeuroAid II (MLC 901)) show an increase in cell survival, an increase in LDH release and an increase of DCX expression in cortical cells. There was no significant difference between the results of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901). Thus, based on the experimental data, it can be demonstrated that both NeuroAid™ (MLC 601) treatments (NeuroAid™ (MLC 601) and NeuroAid II (MLC 901)) enhance neuroproliferation, neurogenesis and neurorepair and may be a form of enhanced treatment for neurological diseases and nervous system injuries.

[0117] The experimental results above also support the use of extracts of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) for improvement of connectivity and survival of all types of neuronal cultures. These extracts could be systematically added to classical culture media for neuronal cultures to considerably improve viability, dendritic growth and synaptogenesis. Example 3 also demonstrates that (NeuroAid™ (MLC 601) and NeuroAid II (MLC 901) may be used for promoting neuronal outgrowth and proliferation of neurons in tissues of the central or peripheral nervous systems, for promoting cell growth, for treating patients having a condition selected from the group of depression, psychiatric indications, natural aging and traumatic brain cell death, diminishing the effect of stroke or neurodegeneration in predisposed subjects or subjects at risk of stroke or neurodegeneration, promotin cell growth, for treating patients with diseased or injured tissues of the central or peripheral nervous systems, for use as a cell culture reagent and for a method for inducing cell survival, growth, proliferation or differentiation of cells.

Example 4

The Effect of NeuroAid II (MLC 901) Pretreatment in Mice 24 h Post-Ischemia

[0118] In this study, mice (n=11) were pretreated for six weeks with a drinking solution of NeuroAid II (MLC 901) and the rate of survival was analyzed at 24 h post-ischemia (FIG. 7A). Control mice (n=10) received a tap water drinking solution.

[0119] To determine whether a pretreatment of NeuroAid II (MLC 901) increases the rate of survival of mice subjected to ischemia, the animals were treated with NeuroAid II (MLC 901) during six weeks before the induction of ischemia. FIG. 8 shows that a six week-pretreatment of NeuroAid II (MLC 901), given in the drinking induced a strong reduction of the mortality of NeuroAid II (MLC 901)-treated animals, compared to control mice. The NeuroAid II (MLC 901) pretreatment during six weeks induced a survival rate of 90.9% as compared to 40.0% in control group.

[0120] Example 4 demonstrates that NeuroAid is beneficial before an ischemic stroke. These studies illustrates that NeuroAid advantageously reduced the infarct size and mortality rate of ischemic mice in a clinically relevant model of stroke.

Example 5

The Effect of Acute NeuroAid II (MLC 901) Post-Treatment in Mice 24 h Post-Ischemia

[0121] In this study, mice (n=10) received an acute post-treatment intraperitoneal injection of NeuroAid II (MLC 901), given at the onset of ischemia and 6 h after reperfusion. Control mice (n=10) was injected with saline solution. The survival rate and the infarct volumes were quantified at 24 h of reperfusion.

[0122] To determine whether acute administration of NeuroAid II (MLC 901) protects against ischemic stroke, mice (n=10) were subjected to ischemia and injected intraperitonally with a single dose of 1 mg/ml of NeuroAid II (MLC 901) solution at the onset of ischemia and 6 h after reperfusion. Acute administration of NeuroAid II (MLC 901) induced a survival rate of 90.0% compared to 38.8% observed in control mice (FIG. 9A). This NeuroAid II (MLC 901) post-treatment drastically decreased cerebral infarction as shown in FIG. 9B. The NeuroAid post-treatment reduced the stroke volume by 47.4% (P<0.001) as compared to control mice.

[0123] These studies demonstrate that NeuroAid is beneficial both before and after an ischemic stroke. These studies illustrates that NeuroAid advantageously reduced the infarct size and mortality rate of ischemic mice in a clinically relevant model of stroke.

Methods for Examples 6 to 8

A. Cortical Cell Culture

[0124] Time-pregnant (E14) C57BI6/J mice were anesthetized with isopentane followed by cervical dislocation. Fetuses were removed and placed in cold (Hanks Balanced Salt Solution) HBSS+solution. Cerebral cortices were dissected in cold HBSS+solution and the meninges were removed. The cortical samples were cut in small pieces and were gently triturated with a fire-polished glass Pasteur pipette in 8 nil HBSS+solution. The mix was filtered (40 μM filter) and centrifuged at 800 rpm for 8 min. The supernatant was removed and the pellet was dissolved in 2 ml culture medium. Cells were plated on Polylysine-coated 12 well (24 mm diameter; Sigma-Aldrich Chimie, St. Quentin Fallavier, France)) plates with glass coverslips (12 mm diameter; CML, Nemours, France) at a density of 1×10.sup.6 cells/well. Cultures were maintained at 37° C. in a humidified 5% CO.sub.2 atmosphere incubator in Neurobasal supplemented with B27, Glutamax, antibiotics were used for experiments after 16 days. Glial growth was suppressed by addition of 5-Fluoro-2-deoxyuridine (2 μM) and Uridine (2 μM) during the second day of culture.

[0125] Experiments were monitored by one researcher blinded to the treatment status (n=3 cultures, 36 wells per experimental group).

B. Cell Injury Assay: Cell Survival and Lactate Dehydrogenase (LDH) Measurements

[0126] Cell viability was assessed at Day 12 of cell culture by using the Cell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay (Promega, Charbonnières-les-Bains, France) (n=3 cultures, 36 wells per experimental group). The assay was a colorimetric method, which was based on the use of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS), a marker of mitochondrial activity and an electron-coupling reagent (phenazine ethosulfate, PES). The MTS tetrazolium compound was bioreduced by cells into a colored formazan product that is soluble in tissue culture medium.

[0127] At Day 12, the totality of cell culture medium was removed and replaced by 500 μl of Neurobasal medium+Cell Titer 96 Aqueous One Solution kit: Cell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay Kit. According to the Proliferation Assay Kit protocol, cells were incubated for 4 hours at 37° C. in the humidified 5% CO2 atmosphere incubator. The reaction was stopped with 2% Sodium Dodecyl Sulfate (SDS). Optical density was measured 4 hours later at 490 nm utilizing a Labsystem Multiscan microplate reader (Labsystem Multiscan RC, VWR International, Fontenay sous Bois, France). Background absorbance at 620 was subtracted. Results were expressed in Optical Density (OD×10-3) representating the number of viable cells. Data was expressed as the percentage of cell viability, which was calculated by dividing the absorbance value of treated samples by that of the untreated control within each group.

[0128] Neuronal injury was quantitatively assessed by the measurement of lactic dehydrogenase (LDH) release from cultured neurons incubated in cell culture mediu at Day 12 of cell culture (Koh and Choi, 1987). The LDH release assay provided a measure of cytoplasmic membrane integrity. At Day 12, 100 μl of the cell culture medium was transferred from culture wells to 96-well plates and mixed with 100 μl of reaction solution according to LDH assay kit (Roche Diagnostic: Cytotoxicity Detection kit: ref 1644793, Indianapolis, USA). Optical density (OD) was measured 30 minutes later at 492 nm utilizing a Labsystem Multiscan microplate reader (Labsystem Multiscan RC, VWR International, Fontenay sous Bois, France). Background absorbance at 620 is subtracted. Neurons exposed to a lysis solution (PBS containing 0.1% Triton X-100) were used as positive control and set as 100% LDH release. Data was expressed as the ratio of LDH efflux/cell viability.

[0129] Results corresponded to the mean of three independent experiments with triplicate determination. Statistical analyses of cell viability and LDH results were assessed using one factor (analysis of variance) ANOVA test following by post-hoc test (P<0.05).

C. Focal Ischemia

[0130] The researchers, who carried out the ischemic surgery and measured infarct volumes were double-blinded in regard to the treatment code.

Model of Focal Ischmia

[0131] Focal ischemia was induced on Adult male C57/B16 mice, weighing 22 to 26 g (7-9 weeks old) by occlusion of the left middle cerebral artery (MCA) using an intraluminal filament technique (Huang et al., 1994) The left common and external carotid arteries were isolated and ligated with a silk 4-0 silk suture (Ethicon). A temporary yasargil aneurysm clip (BMH31, Aesculap, Tuttlingen, Germany) was temporarily placed on the internal carotid artery. A 6-0 coated filament (Doccol, Redlands, Calif., USA), was introduced through a small incision into the common carotid artery and 13 mm distal to the carotid bifurcation for occlusion of the origin of the MCA. Animals were kept at 37° C. for one hour, after which time, the thread was carefully withdrawn to allow reperfusion of the MCA territory. To control the MCAO severity regional CBF (rCBF) was determined by laser-Doppler flowmetry (Perimed) using a flexible 0.5-mm fiber optic extension to the masterprobe fixed on the intact skull over the ischemic cortex (2 mm posterior and 6 mm lateral from the bregma). Sham-operation was performed inserting the thread into the common carotid artery without advancing it to occlude the MCA. The animals were allowed to regain full consciousness on a heating pad before returning to the cage.

Determination of Infarct Volume

[0132] At 30 hours after reperfusion, cresyl violet staining on coronal frozen brain sections (10 μm-thick) was performed using a solution of 1% cresyl violet in 0.25% acetic acid and mounted with Entellan. The striatal and cortical areas of infarction, outlined in light were measured on each section using a computer image analysis system and corrected for brain edema according to Golanov and Reis (Golanov and Reis, 1995). Infarct volume, expressed in mm.sup.3 was calculated by a linear integration of the corrected lesions areas as previously described (Heurteaux et al., 2006a).

D. Drug Treatments

[0133] The composition of NeuroAid II (MLC 901) used (0.4 g per capsule) was as follows:

[0134] 0.57 g Radix astragali,

[0135] 0.114 g Radix salvia miltiorrhizae,

[0136] 0.114 g Radix paeoniae rubra,

[0137] 0.114 g Rhizoma chuanxiong,

[0138] 0.114 g Radix angelicae sinensis,

[0139] 0.114 g Carthamus tinctorius,

[0140] 0.114 g Prunus persica,

[0141] 0.114 g Radix polygalae,

[0142] 0.114 g Rhizoma acori tatarinowii,

[0143] 0.0665 Hirudo.

[0144] For in vitro experiments, cell treatment with NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis were at the concentration of 1 μg/ml, starting at Day 3 of culture during 12 days (corresponding to 15 days of culture).

[0145] For in vivo post-treatment, mice were intraperitonally injected with a single dose of 2 μg/ml NeuroAid II (MLC 901) solution diluted in saline (as vehicle) in a total volume of 500 μl/mouse weighing 25 g, 3 and 24 hours following the end of ischemia. For in vivo pre-treatment, NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis pre-treatment was given in drinking water at the concentration of 6 mg/ml during the 6 weeks before the induction of ischemia.

E. Immunohistochemistry on Cortical Neurons in Culture

[0146] Cortical cells were fixed on coverslips with 4% paraformaldehyde/PBS, permeabilized in 0.3% polyoxyethylensorbitan monolaurate (Tween 20, Sigma) for 10 min and blocked with 2.5% donkey serum in PBS for 2 hours at room temperature. Cells were incubated with a goat anti-doublecortin (DCX) antibody (1:200, SC-8066, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) in 2% donkey serum/phosphate buffer saline overnight (Heurteaux et al., 2006b). After 3 washes in phosphate buffer saline (PBS), cells were incubated in anti-goat Alexa 488-coupled antibodies (FluoProbes, Interchim, Montlucon, France) in 2% donkey serum for 2 hours and washed three times in PBS for 5 minutes each. The neurons were then incubated in Hoechst solution (3 μl in 10 ml, Sigma-Aldricht Chimie, Saint Quentin Fallavier, France) for 10 min to label cell nuclei. After 2 washes in PBS and 1 wash in water, the coverslips were dried and mounted on glass slides with Fluoroprep (75521; Biomérieux, Marcy l′Etoile, France. Cells were observed using epifluorescence microscopy.

[0147] Signal specificity was assessed in negative control coverslips by omitting primary antibody. Epifluorescence microscopy images of protein labeling were captured with identical time of exposition after spectral correction of the autofluorescence background. The differentiated neurites of cortical neurons in culture were observed by DCX immunostaining at Day 12 of treatment. Neurite outgrowth was determined on epifluorescence microscopy by measuring total length of neurites in culture dishes at different times of treatment using a cell photo image and Neurite Tracer Image J software (Pool et al., 2008).

F. Statistical Analyses

[0148] Data was expressed as mean±S.E.M. Statistical analysis of differences between groups was performed by using unpaired t test or ANOVA. Where F ratios were significant, statistical analyses were extended and post-hoc comparisons made by using Tukey's test multiple comparison tests. In all analyses, the level of significance was set at P<0.05.

Example 6

Comparative Effects Between NeuroAid II (MLC 901), Rhizoma Chuanxionq, Radix Angelicae Sinensis and Combined Rhizoma Chuanxionq/Radix Angelicae Sinensis on Cell Viability and LDH Release

[0149] Cortical cells, as prepared by the methods disclosed above, were exposed to a concentration of 1 μg/ml NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis during 12 days.

[0150] The effects of NeuroAid II (MLC 901) to those of Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis treatments against neurodegeneration of cortical cells were compared at Day 15 of culture (Day 12 of treatment) by using cell viability and LDH measurements as disclosed in the methods above. The results are shown in FIG. 10.

Results

[0151] FIG. 10A shows that at the concentration of 1 μg/ml, the three treatments induced a significant increase in neuronal viability as compared to control (*P<0.05, **P<0.01, ***P<0.001) However, NeuroAid II (MLC 901) demonstrated a higher efficacy on cell survival compared to Rhizoma chuanxiong or Radix angelicae sinensis treatments. As shown in FIG. 10A, a NeuroAid II (MLC 901) treatment induced an approximately 51% increase of cell viability, as compared to the control (***P<0.001), while the increase in cell survival induced by Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis was of 37%, 29.5% and 35% respectively. There was a significant difference of efficacy between NeuroAid II (MLC 901) and Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis treatments (##P<0.1) (n=36 wells per group)

[0152] It is well known that increased cell suffering that leads to cell death is associated with increased Lactate DeHydrogenase (LDH) release. FIG. 10B shows that compared to the control, the three treatments significantly reduced the ratio LDH release/cell viability after 12 days of treatment (*P<0.05 and **P<0.01). FIG. 10B also shows that NeuroAid II (MLC 901) significantly reduced LDH release when compared to Rhizoma chuanxiong, Radix angelicae sinensis treatment or combined Rhizoma chuanxiong/Radix angelicae sinensis (##P<0.01) (n=36 wells per group).

[0153] Based on the experimental data in Example 6, it can be demonstrated that NeuroAid II (MLC 901) is more potent in increasing cell viability and reducing LDH release, in culture than either Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis.

Example 7

[0154] Comparative in vivo effects of NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis against ischemic brain injury in vivo

[0155] Post-Treatment: To compare the effects of NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis in vivo, each treatment was tested in a mouse model of focal ischemia.

[0156] Ischemia was induced by transient middle cerebral artery occlusion (MCAO) for 60 min (Huang et al., 1994). Mice (n=12) were subjected to focal ischemia and intraperitoneally injected at 3 hours and then again at 24 hours after MCAO with a single dose of 1 μg of NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis solution.

[0157] Acute administration of each treatment induced a significant survival rate, with a better efficiency obtained with NeuroAid II (MLC 901). FIG. 11A shows that a survival rate of 89% was achieved with NeuroAid II (MLC 901) as compared to a survival rate of 58.5%, 64%, 73% and 75% in ischemic vehicle-, rhizoma chuanxiong-, radix angelicae-treated and combined Rhizoma chuanxiong/Radix angelicae sinensis mice, respectively.

[0158] The drastic decrease of cerebral infarction as shown in FIG. 11B confirms that the best result was observed with NeuroAid II (MLC 901). FIG. 11B shows that NeuroAid II (MLC 901) reduced the stroke volume by 48.4% (′P<0.001) as compared to control ischemic mice at 30 h post-ischemia. The infarct size in rhizoma chuanxiong-, radix angelicae-treated and combined Rhizoma chuanxiong/Radix angelicae sinensis mice was significantly larger than in NeuroAid II (MLC 901)-treated animals after 30 hours of reperfusion (FIG. 11B, #P<0.05, ##P<0.001 versus NeuroAid II (MLC 901) group).

[0159] Pre-treatment: To compare the potential in vivo effects between MLC 901, Rhizoma chuanxiong and Radix angelicae sinensis in prevention against stroke, each treatment was tested in pre-treatment.

[0160] The animals (n=12) were treated with NeuroAid II (MLC 901), Rhizoma chuanxiong or Radix angelicae sinensis administered in the drinking water (6 mg/ml) for 6 weeks before the induction of ischemia. There was no significant difference in the consumption of food and drinking solution between vehicle and NeuroAid II (MLC 901) treated groups. FIG. 12A shows that a 6 week pretreatment of NeuroAid II (MLC 901), Rhizoma chuanxiong and Radix angelicae sinensis induced a reduction of the mortality of treated animals, compared to the control ischemic mice.

[0161] Pre-treatment induced a survival rate of 92%, 78% and 70% in NeuroAid II (MLC 901), Rhizoma chuanxiong and Radix angelicae sinensis, respectively when compared to 70.5% in the control group. The best result for diminishing the effects of stroke or neurodegeneration in predisposed subjects or subjects at the risk of stroke or neurodegeneration obtained with. NeuroAid II (MLC 901) was confirmed by a significant decrease of the infarct volume, which was 39% and 24.5% less important as compared to Rhizoma chuanxiong and Radix angelicae sinensis (FIG. 3B, P<0.01).

[0162] These studies in Example 7 demonstrate that NeuroAid II (MLC 901) is more potent than either Rhizoma chuanxiong or Radix angelicae sinensis in pre-treatment of focal ischemia and NeuroAid II (MLC 901) is more potent than either Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis in post-treatment of focal ischemia.

Example 8

Comparative Effects of NeuroAid II (MLC 901), Rhizoma Chuanxiong, Radix Angelicae Sinensis and Combined Rhizoma Chuanxiong/Radix Angelicae Sinensis on Neuroproliferation and Neurite Outgrowth

[0163] To analyze the effects of NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis on neuronal proliferation and neurite outgrowth, the expression of DCX in cultured cortical cells from embryonic mice was compared after 14 days of treatment.

[0164] Representative epifluorescence microscopy images of DCX staining are shown in FIG. 13. FIG. 13 shows that there was an increase of DCX expression induced by NeuroAid II (MLC 901), Radix angelicae sinensis treatment or combined Rhizoma chuanxiong/Radix angelicae sinensis, highlighting the development of an important axonal and dendritic network with these three treatments as compared to cells treated with the vehicle group or Rhizoma chuanxiong alone.

[0165] Quantification of the fluorescence intensity in each epifluorescence microscopy image was shown in FIG. 14. FIG. 14 showed that the best neuroproliferative effect was obtained in cortical cultures treated with NeuroAid II (MLC 901).

[0166] To investigate whether NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis treatment could promote neurite outgrowth, the total length of neurites in cultured cortical neurons was measured at Day 14 of treatment.

[0167] FIG. 15 shows that NeuroAid II (MLC 901), Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicae sinensis induced a significant neurite outgrowth when compared to vehicle group (*P<0.05, ***P<0.001). The best neurite outgrowth promoting activity was observed for NeuroAid II (MLC 901) when compared to that of Radix angelicae sinensis alone or combined Rhizoma chuanxiong/Radix angelicae sinensis (#P<0.05).

[0168] These studies in Example 8 demonstrate that NeuroAid II (MLC 901) is more potent than either Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicae sinensis for producing neuroproliferation and neurite outgrowth.

Applications

[0169] Advantageously, the disclosed compositions and methods provide a new therapeutic strategy, which focuses on neural repair and restoring neurological functions.

[0170] The disclosed methods provide regenerative therapies for treating nervous system injuries and neurological diseases, by promoting neural growth so as to enable damaged or diseased nerves to function again.

[0171] The disclosed methods can also be used to promote in vitro cell growth. Advantageously, the in vitro culture of various cells may be used for tissue engineering or ex vivo therapeutic uses.

[0172] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.