PROCESS FOR PREPARING A PLATELET LYSATE FRACTION, PLATELET LYSATE FRACTION AND ITS USE FOR TREATING DISORDERS OF THE CENTRAL NERVOUS SYSTEM

Abstract

The present invention relates on a process for preparing platelet lysate fraction, said process comprising the steps of: 1) providing a platelet lysate, 2) collecting from said platelet lysate a fraction wherein the components exhibit a maximum molecular weight of 100 kDa, on a specific platelet lysate fraction and its use as a drug.

Claims

1. A process for preparing a platelet lysate fraction, said process comprising the steps of: 1) providing a platelet lysate, and 2) collecting by ultrafiltration from said platelet lysate a fraction wherein the components exhibit a maximum molecular weight of 100 kDa.

2. The process according to claim 1, wherein the platelet lysate provided in step 1) is a platelet pellet lysate or a pooled human platelet lysate.

3. The process according to claim 1, wherein the collection step is a fractionation carried out by ultrafiltration.

4. The process according to claim 1, wherein the following steps are performed after step 1) and before step 2): a step of heat-treating the platelet lysate at a temperature of about 50° C. to about 70° C. during 15 minutes to 45 minutes, and a step of centrifuging said heat-treated platelet lysate and keeping the supernatant for the collection step.

5. A platelet lysate fraction characterized in that the components contained therein exhibit a maximum molecular weight of 20 kDa.

6. The platelet lysate fraction according to claim 5, characterized in that the components contained therein exhibit a maximum molecular weight of 10 kDa or of 3 kDa, and preferably a maximum molecular weight of 3 kDa.

7. The platelet lysate fraction according to claim 5, having a protein content of less than 1.5 μg/μL, preferably less than 1.0 μg/μL and more preferably less than 0.70 μg/μL.

8. The platelet lysate fraction according to claim 7, wherein the protein content is of about 0.05 μg/μL to about 0.30 μg/μL, and more particularly of about 0.05 μg/μL to about 0.1 μg/μL.

9. The platelet lysate according to claim 8, wherein said fraction is free of fibrinogen.

10. A pharmaceutical composition, comprising the platelet lysate fraction according to claim 5 and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

11. A method for the treatment of disorders of the central nervous system, comprising the step of administering a platelet lysate fraction according to claim 5 to a patient in need thereof.

12. The method according to claim 11, wherein the disorders of central nervous system are selected from neurodegenerative disorders, neurovascular disorders, neuroinflammatory disorders, neurodevelopmental disorders and cerebral insults.

13. The method according to claim 12, wherein the disorders of central nervous system is a neurodegenerative disorder selected from multiple sclerosis (MS), Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), stroke, age-related macular degeneration (AMD), Alzheimer's disease (AD), vascular dementia, frontotemporal dementia, semantic dementia and dementia with Lewy bodies and preferably selected from Parkinson's disease and Amyotrophic lateral sclerosis.

14. The method according to claim 13, wherein the neurodegenerative disorders are selected from Parkinson's disease.

15. The method according to claim 14, wherein the disorder is a cerebral insult selected from hypoxia or traumatic brain injury.

16. The method according to claim 10, wherein the platelet lysate fraction is administrated by intranasal, intrathecal, intraocular or intra cerebroventricular route.

17. The method according to claim 16, wherein the platelet lysate fraction is administrated by intra cerebroventricular route, preferably closed to the intraventricular foramen and more preferably into the third ventricle.

18. The method according to claim 17, wherein said platelet lysate fraction is adapted to be administered with a pump.

Description

FIGURES

[0095] FIG. 1: Time course of treatment with platelet lysate and platelet lysate fractions. Platelet lysate (control) and fractions were added 1 h before erastin (A) or 1 h, 3 h, 6 h and 8 h after erastin (B).

[0096] FIG. 2: Neuroprotective effect measured by cytometry of 50 kDa, H-50 kDa, 30 kDa, H-30 kDa, 10 kDa, H-10 kDa, 3 kDa and H-3 kDa fractions. The viability was measured by propidium iodide assay and normalized to the control (non treated cells) +/−SEM (n=1 for 50 kDa, 30 kDa, 10 kDa and n=2 for 3 kDa).

[0097] FIG. 3: Neuroprotective effect of 50 kDa, H-50 kDa, 30 kDa, H-30 kDa, 10 kDa, and H-10 kDa fractions measured by resazurin. Treatment 1 h before erastin (E). The viability was measured and normalized to the control (non treated cells) n=1.

[0098] FIG. 4: Involved pathway and neuroprotective effect of 3 kDa and H-3 kDa fractions measured by resazurin. Treatment 1 h before erastin (E). iAkt: Akt inhibitor, E: Erastin. The viability was measured and normalized to the control (non treated cells) +/−SEM (n=4).

[0099] FIG. 5: Neuroprotective effect of 3 kDa and H-3 kDa fractions measured by resazurin. Treatment of LUHMES cells 1 h, 3 h, 6 h or 8 h after Erastin (E).

[0100] FIG. 6: Neuroprotective effect measured by cytometric assay of H-3 kDa fraction. Treatment 1 h before Erastin (E). The viability was measured and normalized to the control (non treated cells) +/−SEM (n=4 for pHPL, H-pHPL, H-pHPL-GB, and n=2 for H-3 kDa

[0101] FIG. 7A: Body weight evolution of male mice treated by vehicle and H-PPL diluted preparation. Male WT: Male wild-type, Male Tg: male FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0102] FIG. 7B: Body weight evolution of female mice treated by vehicle and H-PPL diluted preparation. Female WT: Female wild-type, Female Tg: Female FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0103] FIG. 8: Survival curve of male and female mice treated by vehicle and H-PPL diluted preparation. Male Tg: male FVB-Tg (Sod1*G86R), Female Tg: Female FVB-Tg (Sod1*G86R).

[0104] FIG. 9A: Body weight evolution of male mice treated three times a week by vehicle and H-3 kDa preparation. Male WT: Male wild-type, Male Tg: male FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0105] FIG. 9B: Body weight evolution of female mice treated three times a week by vehicle and H-3 kDa preparation. Female WT: Female wild-type, Female Tg: Female FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0106] FIG. 10: Survival curve of male and female mice treated three times a week by vehicle and H-3 kDa preparation. Male Tg: male FVB-Tg (Sod1*G86R), Female Tg: Female FVB-Tg (Sod1*G86R).

[0107] FIG. 11A: Body weight evolution of male mice treated six times a week by vehicle and H-3 kDa preparation. Male WT: Male wild-type, Male Tg: male FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0108] FIG. 11B: Body weight evolution of female mice treated six times a week by vehicle and H-3 kDa preparation. Female WT: Female wild-type, Female Tg: Female FVB-Tg (Sod1*G86R), Veh:Vehicle.

[0109] FIG. 12: Survival curve of male and female mice treated six times a week by vehicle and H-3 kDa preparation. Male Tg: male FVB-Tg (Sod1*G86R), Female Tg: Female FVB-Tg (Sod1*G86R).

EXAMPLES

[0110] The following abbreviations are used throughout the entire description, figures and claims:

[0111] 10 kDa fraction: platelet lysate 10 kDa fraction

[0112] 30 kDa fraction: platelet lysate 30 kDa fraction

[0113] 3 kDa fraction: platelet lysate 3 kDa fraction

[0114] 50 kDa fraction: platelet lysate 50 kDa fraction

[0115] H-10 kDa fraction: heat-treated platelet lysate 10 kDa fraction

[0116] H-30 kDa fraction: heat-treated platelet lysate 30 kDa fraction

[0117] H-3 kDa fraction: heat-treated platelet lysate 3 kDa fraction

[0118] H-50 kDa fraction: heat-treated platelet lysate 50 kDa fraction

[0119] H-pHPL: heat-treated pooled human platelet lysate

[0120] H-pHPL-GB: pooled human platelet lysate mixed with glass beads (GB) and subjected to heat-treatment

[0121] HPL: human platelet lysate

[0122] H-PPL: heat-treated platelet pellet lysate

[0123] ICV: intra cerebro-ventricular

[0124] PAS: platelet additive solution

[0125] PBS: phosphate buffer saline

[0126] PC: platelet concentrate

[0127] pHPL: pooled human platelet lysate

[0128] PL: platelet lysate

[0129] PPL: platelet pellet lysate

[0130] PPL.sup.E: platelet pellet lysate from expired PC

[0131] PPL.sup.F: platelet pellet lysate from non-expired PC

[0132] PRP: platelet-rich plasma

[0133] RBC: red blood cells

Example 1—Experiments with Platelet Pellet Lysate (PPL) as Starting Platelet Lysate Material

[0134] Materials and Methods

[0135] 1. Preparation of Platelet Pellet Lysate and Platelet Lysate Fractions

[0136] Platelet lysates were obtained from platelet concentrates (Etablissement Francais du Sang, Lille, France). After centrifugation at 4600×g for 20 minutes at room temperature, platelet pellets were washed twice and resuspended in PBS in 1/10th of the initial volume. Then, platelet pellets were frozen (nitrogen) and thawed (37° C.) three times, and centrifuged at 4600×g for 20 minutes at room temperature.

[0137] The supernatant, called “Platelet Pellet Lysate” (PPL), was collected, aliquoted and stored at −80° C.

[0138] One part of the PPL was heat-treated at 56° C. for 30 minutes, then centrifuged at 10000×g for 15 minutes at 4° C. and the supernatant, called “Heat-treated Platelet Pellet Lysate” (H-PPL), was aliquoted and stored at −80° C.

[0139] Platelet lysate fractions were obtained from PPL and H-PPL, by performing the fractionation step using Amicon Ultra −0.5 ultrafiltration tubes including different cutoffs (Amicon Ultra-0.5 centrifugal Fitter Devices, Millipore).

[0140] Briefly, 500 μL of PPL or H-PPL is added to the filter device inserted into the collect tube, and centrifuged at 14000×g for 30 minutes at 4° C. with a fixed angle rotor at 40°. According to the cutoff used, the filtrate or the platelet lysate fraction, which is the lower part lower than the cutoff, is called 50 kDa fraction, 30 kDa fraction, 10 kDa fraction and 3 kDa fraction when obtained from PPL, and called H-50 kDa fraction, H-30 kDa fraction, H-10 kDa fraction and H-3 kDa fraction when obtained from H-PPL.

[0141] The different platelet lysate fractions were then aliquoted and stored at −80° C. for further experiments.

[0142] 2. LUHMES Cells Maintenance and Differentiation

[0143] LUHMES cells were obtained from Dr. Scholz's laboratory (University of Konstanz, Germany) and cultured as described.sup.10

[0144] Briefly, undifferentiated LUHMES cells were propagated using Nunclon™ (Nunc, Roskilde, Denmark) plastic cell culture flasks and multi-well plates that were pre-coated with 50 μg/mL poly-L-ornithine and 1 μg/mL fibronectin (Sigma-Aldrich, St. Louis, Mo., USA) in water for 3 h at 37° C. After removal of the coating solution, culture flasks were washed with sterile distilled water and air-dried.

[0145] Cells were grown at 37° C. in a humidified 95% air, 5% CO.sub.2 atmosphere. The proliferation medium was Advanced Dulbecco's modified Eagle's medium (Advanced DMEM)/F12 containing 1× N-2 supplement (Invitrogen, Karlsruhe, Germany), 2 mM L-glutamine (Gibco, Rockville, Md., USA) and 40 ng/mL recombinant bFGF (R&D Systems). When reaching approximately 80% confluence, cells were dissociated with a 0.025% trypsin solution (Gibco, Rockville, Md., USA) and passaged at 3×10.sup.6 cells/flask.

[0146] To induce differentiation intoneuronal cells, 2×10.sup.6 LUHMES were seeded and grown into a T75 flask in proliferation medium for 48 h, then in Advanced DMEM/F12 containing 1× N-2 supplement, 2 mM L-glutamine (Gibco), 1 mM dibutyryl cAMP (Sigma-Aldrich), 1 μg/mL tetracycline (Sigma-Aldrich) and 2 ng/mL recombinant human GDNF (R&D Systems). After two days of culture in differentiation condition, LUHMES were cultured to 24-well plate for further experiments at day six.

[0147] 3. LUHMES Cells Treatment

[0148] All platelet lysate preparations were used at 5% v/v and were tested against cell death induced by Erastin at 1.25 μM. Briefly, LUHMES are used as described before and the different platelet lysate fractions were added into the medium 1 h before treatment with erastin (E) or 1, 3 6 and 8 h after Erastin (FIG. 1).

[0149] 4. Viability Test

[0150] In order to quantify the neuroprotective ability of the different platelet lysate fractions, the viability of LHUMES cells was evaluated by cytometry assay in 24 wells by using propidium iodide incorporation (FIG. 2) and compared to the control or to the different platelet lysates. The cytometer used for the experiments is the CyAn™ model with a 488 nm laser (Beckman Coulter).

[0151] The viability was also measured by a resazurin assay performed in 96 wells at 7 days of differentiation and 24 h after treatments with 50 kDa, H-50 kDa, 30 kDa, H-30 kDa, 10 kDa, and H-10 kDa fractions. The assay is performed on the cell culture without trysinitation (FIG. 3).

[0152] The platelet lysate H-3 kDa fraction was evaluated separately by resazurin assay in order to further determine whether the smallest fraction produced from platelet pellet lysate induces Akt signaling pathway (FIG. 4). Thus, an experiment with Akt inhibitor was performed and the inhibitor MK-2206 was added to the medium at 5 μM 1 h before exposure to platelet lysate fractions.

[0153] The treatment with platelet lysate H-3 kDa fraction was performed 1 h before exposure to Erastin and 1 h, 3 h, 6 h, and 8 h after exposure to Erastin.

[0154] 5. Protein Dosage

[0155] The protein concentration was measured in different samples by a Lowry protein assay. For each sample, measurements were made in duplicate and concentrations are expressed in μg/μL.

[0156] 6. Statistical Analyses

[0157] Results are expressed as the mean±standard error of the mean (SEM). Statistical analyses were performed using one-way ANOVA after checking for the normal distribution of the data. Non-parametric texts of Wilcoxon and Kruskal-Wallis were performed in case of non-normal distribution. A p value of <0.05 was considered statistically significant.

[0158] Results 1. Protein Dosage

TABLE-US-00001 Concentration Standard (μg/μL) Number deviation SEM PPL 17.08 12 4.20 1.21 H-PPL 7.92 14 2.30 0.61 50 kDa fraction 0.52 3 0.21 0.12 H-50 kDa fraction 0.65 3 0.20 0.11 30 kDa fraction 0.36 2 0.16 0.12 H-30 kDa fraction 0.56 2 0.12 0.09 10 kDa fraction 0.41 2 0.22 0.16 H-10 kDa fraction 0.55 3 0.26 0.15 3 kDa fraction 0.36 7 0.09 0.04 H-3 kDa fraction 0.40 13 0.10 0.03

[0159] 2. Protective Ability on Dopaminergic Neurons [0160] Cytometric Assay

[0161] As shown in FIG. 2, the viability of LUHMES cells treated with Erastin declines to approximately 30%. Thus, Erastin kills the control cells and this was not observed when LUHMES cells were treated concomitantly by any of platelet lysate fractions.

[0162] Indeed, none of the platelet lysate fractions had a toxic effect to the LUHMES cells. Therefore, the platelet lysate fractions protect LUHMES cells from death induced by Erastin and display strong neuroprotective effect. [0163] Resazurin Assay

[0164] The results obtained by cytometric assay were confirmed by resazurin.

[0165] As show in FIG. 3 and FIG. 4, Erastin kills efficiently the LUHMES cells and a treatment with platelet lysate fractions according to the invention 1 h before exposure to Erastin protects LUHMES cells from death. Thus, the platelet lysate fractions are able to prevent the toxic effect of Erastin and display significant neuroprotective effect.

[0166] Moreover, FIG. 4 also shown that platelet lysate H-3 kDa fraction involves Akt signaling pathway.

[0167] The neuroprotective effect was also tested when platelet lysate 3 H-kDa fraction was added after exposure to Erastin and the result shown (FIG. 5) that a treatment with platelet lysate H-3 kDa fraction 1 h to 8 h before exposure to Erastin still protects LUHMES cell from death.

[0168] 3. Conclusion

[0169] The platelet lysate fractions according to the invention prepared with platelet pellet lysate as starting material are able to protect cells against death induced by a neurotoxic and display an effective neuroprotective effect. This result was validated with two different assays.

Example 2—Experiments with Pooled Human Platelet Lysate (pHPL) as Starting Platelet Lysate Material

[0170] 1. Preparation of the Pooled Human Platelet Lysate (pHPL) and Platelet Lysate Fractions

[0171] The pooled human platelet lysate (pHPL) is obtained from Macopharma (Tourcoing, France) under the name MultiPL'30® Human platelet lysate, reference BC0190020. A part of the pHPL was subjected to heat-treatment at 56° C. for 30 min and was purified by centrifugation (15 minutes, 10000×g, 4° C.) to obtain the so-called HT-pHPL.

[0172] An another part of the pHPL was mixed with 0.5 g/mL of glass beads (BEAD-002-1 kg of 2 mm of diameter, from Labbox) and CaCl.sub.2 (23 mM final concentration; C4901 Calcium chloride anhydrous powder, from Sigma-Aldrich) under stirring for 1 h. It was leading to a clot formation that was removed after centrifugation (6000×g for 30 minutes at 22° C.). The supernatant was heated at 56° C. for 30 minutes and centrifuged (10000×g for 15 minutes at 4° C.) before aliquots were made and stored at −80° C. for further use. This so-obtained platelet lysate is called H-pHPL-GB.

[0173] The heat-treated. platelet lysate 3 kDa fraction was obtained from H-pHPL-GB using Amicon Ultra −0.5 ultrafiltration tubes including different cutoffs (Amicon Ultra-0.5 centrifugal Filter Devices, Millipore).

[0174] Briefly, 500 μL of H-pHPL-GB is added to the filter device inserted into the collect tube, and centrifuged at 14000×g for 30 minutes at 4° C. with a fixed angle rotor at 40°. According to the cutoff used, the filtrate or the heat-treated platelet lysate fraction, which is the lower part lower than the cutoff, is called H-3 kDa because it was obtained from a heat-treated platelet lysate.

[0175] The platelet lysate fractions were then ali quoted and stored at −80° C. for further experiments.

[0176] 2. LUHMES Cells Maintenance and Differentiation

[0177] LUHMES cells were obtained and prepared as described in example 1.

[0178] 3. LUHMES Cells Treatment

[0179] The H-3 kDa fraction was used at 5% v/v and was tested against cell death induced by Erastin. Briefly, LUHMES are used as described before and the H-3 kDa fraction was added into the medium 1 h before treatment with Erastin (E).

[0180] 4. Viability Test

[0181] In order to quantify the neuroprotective ability of the heat-treated platelet lysate 3 kDa fraction, the viability of LHUMES cells was evaluated by cytometry assay in 24 wells by using propidium iodide incorporation and compared to the control or to the different platelet lysates. The cytometer used for the experiments is the CyAn™ model with a 488 nm laser (Beckman Coulter).

[0182] 5. Protein Dosage

[0183] The protein concentration was measured in different samples by a Lowry protein assay. For each sample, measurements were made in duplicate and concentrations are expressed in μg/μL.

[0184] 6. Results [0185] protein dosage

TABLE-US-00002 Concentration Number of Standard (μg/μL) samples deviation SEM pHPL 19.08 2 2.47 1.75 H-pHPL 18.51 2 1.20 0.85 pHPL-GB 18.65 2 0.62 0.44 H-pHPL-GB 17.31 2 1.58 1.12 H-3 kDa 0.09 2 0.02 0.02 [0186] Protective ability on dopaminergic neurons

[0187] As shown in FIG. 6, the viability of LUHMES cells treated with Erastin declines to approximately 50%. Thus, Erastin effectively kills the control cells and this was not observed when LUHMES cells were treated concomitantly the H-3 kDa fraction.

[0188] Therefore, the H-3 kDa fraction protects LUHMES cells from death by ferroptosis and display strong neuroprotective effect.

[0189] This example exhibits the potential of the heat-treated platelet lysate 3 kDa fraction according to the invention. Moreover, this fraction was obtained from a pooled human platelet lysate which offers a substantially higher safety margin than standard human platelet lysates suspended in plasma. Thus, this H-3 kDa fraction is more suitable and more efficient for use in biotherapy, especially through brain administration.

Example 3—In Vivo Tests

[0190] All experiments were carried out in accordance with the “Principles of Laboratory Animal Care” (NIH publication 86-23, revised in 1985) and the current French and European Union legislative and regulatory framework on animal experimentation (The Council of the European Communities Directive 86/609).

[0191] The mice enrolled were FVB-Tg (Sod1*G86R) M1Jwg/J mice from JAX laboratories. Animals were group-housed (10 per cage) in a temperature-controlled room (22±2° C.) with a 12/12-hour light/dark cycle. Food and water were feed ad-libitum. After reception, the animals had a 7-day habituation period with no handling. Breeding was realized in SOPF facility and genotyping is performed by qPCR (from tail biopsy). Animal are identified with earrings.

[0192] 1. Experimental Protocol

[0193] Mice were handled and weighted at the age of 60 days and they were evaluated twice a week (i.e. body weight and neuroscore) from the age of 67 days to their death.

[0194] a. Neuroscore [0195] walking (0=ok, 1=a single hindpaw alteration, 2=two hindpaws alteration), [0196] tail suspension test (0=ok, 1=a single hindpaw retractation, 2=two hindpaws retractation) [0197] paralysis (0=no, 1=yes) [0198] Kyphosis (0=no, 1=yes)

[0199] Max score=6 (moribund score)

[0200] b. Treatment

[0201] From 75 days to death, different platelet lysate preparations versus vehicle are administrated three times a week (preparations 1 and 2) and six times a week (preparation 2 only) in SOD1m-FVB and WT-FVB males and females. The SOD1m-FVB mice are transgenic mice overexpressing mutant forms of the copper/zinc superoxide dismutase gene. The dose of preparation administrated was 20 μL intra nasally (i.n.).

[0202] c. Platelet Lysate Tested

[0203] Three platelet lysate preparations have been tested and are described hereafter.

[0204] Preparation 1: H-PPL as described in example 1, section 1 and diluted at 50% in PBS (H-PPL diluted).

[0205] Preparation 2: heat-treated platelet lysate 3 kDa fraction (H-3 kDa fraction) prepared from H-PPL as described in example 1, section 1.

[0206] d. Experimental Groups

[0207] Eight groups have been constituted as follow:

[0208] Males WT-FVB+vehicle

[0209] Males WT-FVB+preparations (H-PPL, H-PPL diluted or H-3 kDa fraction)

[0210] Males SOD1m-FVB+vehicle

[0211] Males SOD1m-FVB+preparation (H-PPL, H-PPL diluted or H-3 kDa fraction)

[0212] Females WT-FVB+vehicle

[0213] Females WT-FVB+preparation (H-PPL, H-PPL diluted or H-3 kDa fraction)

[0214] Females SOD1m-FVB+vehicle

[0215] Females SOD1m-FVB+preparation (H-PPL, H-PPL diluted or H-3 kDa fraction)

[0216] 2. Results

[0217] a. Preparation 1: H-PPL Diluted [0218] Body weight

[0219] As shown in FIGS. 7A and 7B, treatments with H-PPL diluted had no effect in WT males and females.

[0220] Regarding to the body weight decline, no difference was observed in Tg males. Nevertheless, in Tg females, H-PPL diluted treatment maintains the body weight at the beginning level up to Day 95 with almost 10 days of difference with vehicle group and also delays the pre-mortem body weight from 10 days (D102-D112). [0221] Survival curve

[0222] In accordance with the body weight, in Tg females treated with H-PPL diluted had an extended survival from D105 to D116 (11 days more). This difference was not observed in Tg Male mice between the vehicle and treated group. (FIG. 8)

[0223] b. Preparation 2: H-3 kDa Fraction

[0224] Three Times a Week Administration: [0225] Body weight

[0226] As shown in FIG. 9A, in the WT mice, H-3 kDa fraction had no effect in male body weight. Contrary to the males, in females a slight decrease is observed shortly after the beginning of the treatment (D81) and throughout its duration.

[0227] In both Tg males and females, no improvement is observed in body weight loss initiation (compare to Tg mice with vehicle) but, H-3 kDa fraction induced an important delay in the pre-mortem body weight of 21 days for females mice (from D105 to D126) and of 7 days for males mice (from D112 to D119) (FIG. 9B). [0228] Survival curve

[0229] As show in FIG. 10 and in accordance with the delay observed in the post-mortem body weight, a treatment with H-3 kDa fraction extended survival time up to 21 days for Tg females (D109 to D130) and up to 7 days for Tg males (D116 to D123).

[0230] Six Times a Week Administration: [0231] Body weight

[0232] As shown in FIGS. 11A and 11B, in the WT mice, H-3 kDa fraction had no effect in male and female body weight.

[0233] In both Tg males and females, no improvement is observed in body weight loss initiation (compare to Tg mice with vehicle) but, H-3 kDa fraction induced a delay in the pre-mortem body weight of 11 days for males mice (from D119 to D130). This effect is not observed in Tg Female. [0234] Survival curve

[0235] As show in FIG. 12, and in accordance with the delay observed in the post-mortem body weight, a treatment with H-3 kDa fraction extended survival time up to 21 days for Tg females (D109 to D130) and up to 7 days for Tg males (D116 to D123).

[0236] 3. Conclusion

[0237] No irritation was observed with H-PPL diluted and H-3 kDa treatments.

[0238] Regarding the survival curves obtained with different preparation, it can be observed that the effect depends on animal gender.

[0239] But, the main conclusion of this in vivo test is that H-3 kDa has an excellent safety profile and a great efficacy with a classical sex-dose related effect. Indeed, treatment with H-3 kDa extended survival by 7 days in Tg males mice and provided an increase of about 21 days in Tg females mice (90% of improvement as compared with controls) and of about 10 days in Tg males mice (48% of improvement as compared with controls). Those results show the potential of the heat-treated platelet lysate 3 kDa fraction according to the invention in order to induce neuroprotective ability.

REFERENCES

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