Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/19—Platelets; Megacaryocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0644—Platelets; Megakaryocytes

Definitions

• the present invention relates to a process for obtaining a platelet lysate fraction, the platelet lysate fraction itself and its use for treating disorders of the central nervous system such as neurodegenerative, neuroinflammatory, neurodevelopmental and/or neurovascular disorders (i.e strokes), but also the consequences of cerebral insults such as traumatic brain injury or hypoxia.
• Developing effective "disease modifying strategy" providing neuroprotection, neurorestoration and neurogenesis to treat neurodegenerative disorders, such as Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Alzheimer disease (AD), is urgently needed considering the huge societal and economic impacts these disorders impose to patients and care-givers.
• neurotrophins as activators and modulators of neuronal signaling pathways, represent a logical therapeutic strategy for neurological disorders.
• Application of single recombinant neurotrophic growth factors has provided encouraging results for neuronal protection and repair in both cell and animal models.
• Platelet-derived growth factor-CC proved to be a potent neuroprotective factor in several animal models of neuronal injury whereas PDGF-BB and brain-derived neurotrophic factor (BDNF), administered via intra cerebro- ventricular (ICV) route, stimulated neurogenesis.
• BDNF brain-derived neurotrophic factor
• ICV intra cerebro- ventricular
• TGF- ⁇ Transforming growth factor- ⁇
• TGF- ⁇ Transforming growth factor- ⁇
• b-FGF basic-fibroblast growth factor
• VEGF- ⁇ vascular endothelial growth factor- ⁇
• Platelet concentrates are well-established therapeutic product, on the WHO model list of essential medicines, typically used in the prophylaxis and treatment of bleeding disorders resulting from thrombocytopenia. Besides their role in haemostasis, platelets exert crucial physiological functions in wound healing and tissue repair.
• the range of regenerative medicine 3 and cell therapy 4 applications where platelets and platelet lysates are evaluated is expanding.
• the therapeutic benefit of platelets in tissue healing is multifactorial and results from the myriad of bioactive mediators stored primarily in the a- granules and acting in synergy.
• bioactive mediators stored primarily in the a- granules and acting in synergy.
• bioactive mediators stored primarily in the a- granules and acting in synergy.
• bioactive mediators stored primarily in the a- granules and acting in synergy.
• bioactive mediators stored primarily in the a- granules and acting in synergy.
• bioactive mediators stored primarily in the a- granules and acting in synergy.
• platelet lysates Intracranial delivery of platelet lysates in animal models of stroke was recently shown to stimulate the proliferation of endogenous neural stem cells (eNSC) and angiogenesis in the subventricular zone and in the peri-lesion cortex, leading to improved functional outcomes and reduced injury, and suggesting neuroprotective effects 5 .
• eNSC endogenous neural stem cells
• angiogenesis in the subventricular zone and in the peri-lesion cortex
• platelet lysates contain a huge pool of molecules and compounds which are not fully characterized, but it is known that platelet lysate contains plasma-borne fibrinogen, a protein that plays a causative role in neurologic disorders as a potent inducer of inflammation and an inhibitor of neurite outgrowth.
• the present invention relates to a process for preparing a platelet lysate fraction, said process comprising the steps of:
• the first step of the process consists in providing a platelet lysate.
• This platelet lysate may be a platelet pellet lysate (PPL) or a pooled human platelet lysate (pHPL).
• PPL platelet pellet lysate
• PHPL pooled human platelet lysate
• the platelet lysate is a pooled human platelet lysate (pHPL).
• Both PPL and pHPL may be prepared according to well-known methods from platelet concentrate (PC), which induce the release of growth factors and other active molecules.
• PC platelet concentrate
• the platelet lysate provided in step 1) is a platelet pellet lysate (PPL).
• PPL may be prepared as described in the art 6 . It may for example be prepared as follows: i) Providing a platelet concentrate (PC), ii) Centrifuging said platelet concentrate so as to obtain a platelet pellet and a first supernatant, iii) Removing the supernatant and suspending the pellet in a physiological buffer, iv) Freeze-thawing said suspended pellet, v) Centrifuging the suspension obtained in step iv) so as to obtain a platelet pellet lysate and second supernatant.
• PC platelet concentrate
• iii) Centrifuging said platelet concentrate so as to obtain a platelet pellet and a first supernatant
• iii) Removing the supernatant and suspending the pellet in a physiological buffer iv) Freeze-thawing said suspended pellet
• the platelet concentrate provided in step i) may be obtained by suitable standard collection methods from autologous or allogeneic platelet sources, in particular from whole blood, or by apheresis procedures, and suspended in plasma, or a combination of plasma and platelet additive solution, or platelet additive solution only . Moreover, the platelet concentrate may be leukoreduced.
• Suitable physiological buffers used in step iii) are for example phosphate buffer saline (PBS), HEPES buffer, Tris-HCl buffer or sodium acetate buffer, or physiological saline.
• the platelet pellet lysate may be fresh PPL (PPL F ) or expired PPL (PPL E ), preferably PPL .
• fresh PPL refers to platelet pellet lysate prepared from platelet concentrates processed within 5 days of collection (non-expired).
• expired PPL refers to platelet pellet lysate prepared from platelet concentrates processed over 5 days of storage.
• the platelet lysate provided in step 1) is a pooled human platelet lysate (pHPL).
• the pHPL may be prepared by the method comprising the following steps of:
• step c) mixing the lysates resulting from step b) in order to obtain a pooled human platelet lysate.
• the platelet concentrates provided in step a) may come from different donors and may be obtained by suitable standard collection methods from allogeneic platelet sources.
• the platelet concentrate may be obtained from whole blood using the buffy coat or platelet-rich plasma (PRP) technique, or may be collected by apheresis technique.
• the platelet concentrate is produced from whole blood using the buffy coat or (PRP) technique 8 .
• PRP method anticoagulated whole blood is centrifuged using a soft spin under conditions validated to segregate red blood cells (RBC) from the upper half containing a platelet and plasma mixture, so called PRP. Platelets are then concentrated by hard spin centrifugation with validated acceleration and deceleration curves.
• RBC red blood cells
• the platelet concentrate bag is left stationary at room temperature and then the concentrate is resuspended in plasma.
• anticoagulated whole blood is centrifuged using a hard spin with validated acceleration and deceleration curves to separate 'cell-free" plasma on the top layer, a middle layer called buffy coat (BC) and a red blood cells (RBC) bottom layer.
• the BC layer is transferred to a satellite bag.
• a small quantity of plasma is returned to the BC layer and gently mixed before again being subjected to light spinning centrifugation with validated acceleration and deceleration curves.
• the PRP supernatant is then placed in platelet storage and may be stored at 22+/- 2°C.
• the platelet concentrates may be obtained through an extracorporeal medical device used in blood donation that separates the platelets and returns other portions of the blood to the donor.
• the plasma used for suspending the concentrate in the "PRP method", the plasma returned to BC layer in the "buffy coat” method, or the plasma collected with platelet by apheresis may be substituted by a platelet additive solution (PAS) or by a mixture between plasma and PAS, and preferably by a mixture between plasma and PAS.
• PES platelet additive solution
• Said mixture between plasma and PAS may contain from about 30% to 40% by weight of plasma and from about 70% to 60% by weight of PAS.
• the platelet concentrate provided in step a) may be subjected to a leucodepletion treatment. This treatment leads to leucocyte depletion and it may be achieved by filtration on a leucoreduction filter or during the platelet collection by apheresis.
• the platelet concentrate provided in step a) may be subjected to a step of viral/pathogen inactivation treatment before lysis.
• the viral/pathogen inactivation treatment applied to the platelet concentrate may be selected from Intercept® Blood system (from Cerus Corporation), Mirasol® PRT system (from Terumo BCT), or THERAFLEX-UV (from Macopharma). These procedures are well-known by one skilled in the art and target, with or without the addition of a photo-inactivating agent, the alteration of nucleic acids.
• the platelet concentrate may also be subjected to a leucodepletion treatment and to a viral/pathogen inactivation treatment.
• the leucodepletion treatment is performed before the viral/pathogen inactivation treatment.
• the step b) of lysing separately each platelet concentrates may be achieved by any method known in the art.
• platelet lysis may be achieved by one or more freeze/thaw cycles, by platelet activation induced by addition of thrombin or CaCl 2 , by sonication or by solvent/detergent (S/D) treatment.
• step b) of lysis is achieved by one or more freeze/thaw cycles, and more preferably by at least three cycles.
• a centrifugation and filtration step may also be performed to remove cell debris.
• step c) consists in mixing the lysates in order to obtain a pooled human platelet lysate, also called pHPL.
• the pool of HPL is obtained by mixing the lysed platelet concentrates from at least 2 platelet lysates from different donors.
• the pool of HPL is obtained by mixing at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, , at least 140, at least 180, at least 200 and more particularly, at least 240 different platelet lysates collected from different donors.
• a suitable pooled human platelet lysate (pHPL) for the process of the invention may be any pooled human platelet lysate from blood establishments or from commercial suppliers.
• the pooled human platelet lysate may be obtained from Macopharma (Tourcoing, France; MultiPL'30® Human platelet lysate), from Cook-Regentec (Indianapolis, USA; Stemulate ® Human platelet lysate), from Stemcell Technologies (Grenoble, France; Human platelet Lysate) or also from Sigma- Aldrich (PLTMax® Human Platelet Lysate).
• the pHPL may be subjected to a treatment which induces an activation of the coagulation cascade.
• the pHPL may be mixed with glass beads (GB) and CaCl 2 under stirring, or using CaCl 2 alone.
• This treatment leads to a clot formation that is removed after centrifugation and the resulting pHPL is thus free of fibrinogen.
• the inventors believe that this treatment contributes to lower toxicity and improved neuroprotective effect of the obtained platelet lysate fraction according to the invention.
• the second step of the process consists in collecting a platelet lysate fraction wherein the components exhibit a maximum molecular weight of 100 kDa.
• the collected heat- treated platelet lysate fraction may not contain components with a molecular weight greater than 100 kDa.
• a platelet lysate fraction wherein the components exhibit a maximum molecular weight of X kDa is called "platelet lysate X kDa fraction" or also "X kDa fraction”, e.g "platelet lysate 100 kDa fraction" or "100 kDa fraction”.
• the collection step is performed in order to obtain a platelet lysate fraction wherein components exhibit a maximum molecular weight of 100 kDa, of 90 kDa, of 80 kDa, of 70 kDa, of 60 kDa, of 50 kDa, of 40 kDa, of 30 kDa, of 20 kDa, of 10 kDa, of 5 kDa, of 3 kDa or of 1 kDa.
• the platelet lysate fraction may not contain components with a molecular weight greater than 100 kDa, 90 kDa, 80 kDa, 70 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa, 5 kDa, 3 kDa, or greater than 1 kDa.
• the so-obtained fractions are preferably the platelet lysate 50 kDa fraction, the platelet lysate 30 kDa fraction, the platelet lysate 20 kDa fraction, the platelet lysate 10 kDa fraction and the platelet lysate 3 kDa fraction, more preferably the platelet lysate 10 kDa fraction and the platelet lysate 3 kDa fraction, and even more preferably the platelet lysate 3 kDa fraction.
• the platelet lysate fraction according to the invention exhibits a strong neuroprotective activity although the platelet lysate was subjected to a collection step in order to remove components. Indeed, the so- obtained fractions still exert a neuroprotective effect while it was expected that the removing of some components according to their molecular weight would lead to the loss of said effect. Still surprisingly, the neuroprotective activity is obtained for the smallest fractions for which it was believed that the lack of components with high molecular weight would have been detrimental for the neuroprotective activity.
• the present invention represents a major breakthrough to provide an alternative treatment of central nervous system disorders.
• the collection step may be performed by any method known in the art which leads to the separation and/or concentration of components contained in a liquid according to their molecular weight.
• the collection step may consist in fractionating the supernatant obtained by centrifugation of the platelet lysate in order to collect the so-called platelet lysate 100 kDa fraction.
• the fractionation may be performed by ultrafiltration.
• a centrifugal filter with vertical membrane having a 100 kDa cut-off may be used.
• the centrifugal filter is thus filled with the supernatant obtained after the third step and is subjected to the centrifugation.
• the angle rotor, the spin speed and the spin time may be determined by one skilled in the art.
• one skilled in the art may adapt the cut-off used in order to obtained the desired platelet lysate fraction according to the invention.
• the platelet lysate fraction of the invention has a reduced protein content.
• reduced protein content means that said fraction contains less than 1.5 ⁇ g/ ⁇ L of proteins, preferably less than 1.0 ⁇ g/ ⁇ L, and more preferably less than 0.70 ⁇ g/ ⁇ L.
• the protein content may be determined by any method known in the art and for example by Lowry protein assay or by ELISA.
• the platelet lysate 3 kDa fraction may present a protein content of about 0.05 ⁇ g/ ⁇ L to about 0.30 ⁇ g/ ⁇ L, particularly of about 0.05 ⁇ g/ ⁇ L to about 0.30 ⁇ g/ ⁇ L, and more particularly a protein content of about 0.05 ⁇ g/ ⁇ L to about 0.1 ⁇ g/ ⁇ L.
• the platelet lysate 3 kDa fraction is preferably free of fibrinogen and free of growth factors.
• the process of the invention may comprise an additional step of storing the heat-treated platelet lysate fraction at -80°C for further use.
• the process of the invention may also comprises 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.
• the heat-treatment step is preferably performed without adding stabilizers that are classically used to maintain the biologic activity of protein.
• stabilizers are for examples sucrose, sorbitol, mannitol or amino acids such as arginine or lysine.
• the heat-treatment step induces precipitation of some proteins which are thus removed after the centrifuging step.
• a reduced content in protein may be more advantageous for some applications, for example in the treatment of a disorder of the central nervous system through an intranasal administration.
• the heat-treatment step may be performed at a temperature of about 50°C to about 70°C, preferably at a temperature of about 50°C to about 60°C, and more preferably at a temperature of about 54°C to 58°C.
• the heat-treatment step is for example performed at 56°C.
• the duration of the heat- treatment step may be from 15 to 45 min, preferably from 20 to 40 minutes, and more preferably from 25 to 35 minutes. For example, the heat-treatment step is performed for 30 minutes.
• the centrifugation may advantageously be carried out at a temperature of about 2°C to 6°C.
• the duration of this centrifugation step is at least 10 minutes and the speed may be of about 8000 x g to about 12000 x g, preferably of about 9000 x g to about 11000 x g, and more preferably around 10000 x g.
• the supernatant is recovered and used for the collection step of the process.
• the resulting platelet lysate fraction after the collection step is a heat-treated platelet lysate fraction. It has been surprisingly and unexpectedly found that heat-treated platelet lysate fractions according to this embodiment exhibit a strong effect in terms of neuroprotection although the platelet lysate was subjected to a heat-treatment step and to a collection step.
• the fractions prepared according to the process of the invention protect dopaminergic cells from death induced by a neurotoxin.
• the improved neuroprotective activity of the fractions is a result of their reduced protein content, such as fibrinogen content, and the result of the concentration of compounds with a molecular weight not greater than 100 kDa, and particularly, not greater than 50 kDa, 30 kDa, 20 kDa, 10 kDa or 3 kDa.
• ALS amyotrophic lateral sclerosis
• the collection step, and optionally the heat-treatment result in reduction or depletion of fibrinogen and proteolytic enzymes (such as thrombin, or thrombin-like, or thrombin-generating coagulation factors), and particularly that the heat- treatment step precipitates and/or inactivates potentially toxic heat-unstable proteins and favorably modifies the protein and growth factor balance in the resulting fraction as well as the collection which modifies the component's molecular weight balance and potentiate the neuroprotective effect.
• the resulting platelet lysate fraction may avoid the biological risk of fibrin formation, which is toxic for the brain.
• the obtained platelet lysate fraction according to the invention offers a substantially higher safety margin than standard human platelet lysates suspended in plasma and is more suitable and more efficient for use in biotherapy, especially through brain administration.
• platelet lysate fractions obtained by the process of the invention provides improved neuroprotective activity.
• the invention in a second aspect, relates to a platelet lysate fraction.
• the platelet lysate fraction may be obtained according to the method described above.
• the platelet lysate fraction according to the invention is specifically a platelet fraction wherein the components exhibit a maximum molecular weight of 100 kDa.
• the platelet lysate fraction according to the invention is a platelet lysate 50 kDa fraction, a platelet lysate 30 kDa fraction, a platelet lysate 20 kDa fraction, a platelet lysate 10 kDa fraction or a platelet lysate 3 kDa fraction.
• the fraction according to the invention is a platelet lysate 10 kDa fraction or a platelet lysate 3 kDa fraction, and even more preferably a heat-treated platelet lysate 3 kDa fraction.
• the platelet lysate fraction of the invention has a reduced content in proteins.
• reduced content it is meant that said fraction contains less than 1.5 ⁇ g/ ⁇ L of proteins, preferably less than 1.0 ⁇ g/ ⁇ L and more preferably less than 0.70 ⁇ g/ ⁇ L.
• the platelet lysate 3 kDa fraction may present a protein content of about 0.05 ⁇ g ⁇ L to about 0.30 ⁇ g/ ⁇ L, particularly of about 0.05 ⁇ g/ ⁇ L to about 0.30 ⁇ g/ ⁇ L, and more particularly a protein content of about 0.05 ⁇ g/ ⁇ L to about 0.1 ⁇ g/ ⁇ L.
• the heat- treated platelet lysate 3 kDa fraction is preferably free of fibrinogen.
• the platelet lysate fraction may be obtained by the process described hereabove.
• the platelet lysate fractions according to the invention display a strong neuroprotective activity and are particularly advantageous for treating disorders of the central nervous system. More particularly, the smallest platelet lysate fractions, i.e the platelet lysate 10 kDa fraction and the platelet lysate 3 kDa fraction, can more easily pass through the nasal cavity in order to penetrate into the brain and exert the neuroprotective effect.
• the invention relates to the platelet lysate fraction according to the invention for use as a biological drug or "biotherapy”.
• platelet lysate fraction may be used in the treatment and/or prevention of a disorder of the central nervous system.
• the invention also relates to a method of treating and/or preventing disorders of the central nervous system, comprising the administration of a therapeutically effective amount of the platelet lysate fraction of the invention, to a patient in need thereof.
• the patient is a warm-blooded animal, more preferably a human.
• disorders of the central nervous system within the meaning of present invention include but are not limited to neurodegenerative disorders, neurovascular disorders, neuroinflammatory disorders, neurodevelopmental disorders such as autism and schizophrenia, cerebral insult such as severe hypoxia following delivery or cardiac arrest or severe cranial traumatism/traumatic brain injury that is to say severe insults resulting in a significant loss of neurons leading to handicap.
• the disorder of the central nervous system is a neurodegenerative disorder.
• Neurodegenerative disorders within the meaning of the present invention include, but are not limited to multiple sclerosis (MS), Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), stroke, age-related macular degeneration (AMD), degenerative diseases of the retina, and dementia, the latter including, without being limited thereto, Alzheimer's disease (AD), vascular dementia, fronto temporal dementia, semantic dementia and dementia with Lewy bodies.
• the neurodegenerative disorder is selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and Amyotrophic lateral sclerosis, more preferably from Parkinson's disease and Amyotrophic lateral sclerosis.
• the disorder of the central nervous system is a cerebral insult of the central nervous system such as severe hypoxia following delivery or cardiac arrest or severe cranial traumatism that is to say severe insults resulting in a significant loss of neurons leading to handicap.
• a cerebral insult of the central nervous system such as severe hypoxia following delivery or cardiac arrest or severe cranial traumatism that is to say severe insults resulting in a significant loss of neurons leading to handicap.
• the early treatment, with the platelet lysate fraction, following the insult may enhance the physiological neurorestoration and neurogenesis abilities.
• the platelet lysate fraction may be administered as such, be encapsulated in natural or synthetic nanoparticles 9 or microparticles or be comprised in a pharmaceutical solution further comprising at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
• the pharmaceutical solution can further comprise complexes, molecules, peptides, salts, vectors or any other compound, which can ameliorate or can be beneficial in treatment neurological disorders.
• the route of administration and the dosage regimen naturally depend upon the severity of the illness, the age, weight, and sex of the patient, etc.
• the platelet lysate fraction of the invention may be used for the treatment of any patient, especially a warm-blooded animal such as a mammal and preferably a human.
• the platelet lysate fraction according to the invention is suitable for the administration in the central nervous system.
• said platelet lysate fraction is adapted for intranasal (e.g. Parkinson's disease which is a pathology of the substantia nigra, striatum and olfactory bulbar close to the nasal cavities) or intra thecal (e.g. for amyotrophic lateral sclerosis which is a pathology of the spinal cord) or intra cerebroventricular (ICV) administration, preferably closed to the intraventricular foramen so that the platelet lysate fraction can be administrated into the third ventricle.
• Parkinson's disease which is a pathology of the substantia nigra, striatum and olfactory bulbar close to the nasal cavities
• intra thecal e.g. for amyotrophic lateral sclerosis which is a pathology of the spinal cord
• intra cerebroventricular (ICV) administration preferably closed to the intraventricular foramen so that
• the smallest platelet lysate fractions still exhibit a neuroprotective activity, they are particularly effective for an intranasal administration. Indeed, thanks to their low molecular weight components, those fractions can easily penetrate the brain through the nasal cavity which is advantageous for the purpose of the invention in order to treat disorders of the central nervous system. Moreover, in terms of safety, it is more advantageous for the patient to get benefit from neuroprotective effect with a platelet lysate which contain less components.
• Administration to the central nervous system may be achieved by the method known in the art.
• administration may be carried out with a drug delivery system, such as a programmable medication pump.
• the administration of the platelet lysate fraction of the invention may also be performed by any other method known by the person skilled in the art, such as for example, intraveneous, intraperitoneal, intramuscular or intraocular administration, or perfusion or infusion of an organ (i.e. direct infusion of a part of the brain tissue).
• the exposure dosage used for the administration may be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology or of the desired duration of treatment.
• neuroprotective activity or “neuroprotection” is meant preservation of neuronal structure and/or function of neuronal cells affected by neurotoxin compared to neuronal cells, which are not affected by neurotoxin.
• Neuroprotection aims to prevent or slow the disease progression and secondary injuries by halting or at least slowing the loss of neurons. For example, it refers to preservation of the number of neurons in the striatum and/or in the substantia nigra pars compacta of patients affected by Parkinson's disease compared to patients who are not affected by Parkinson's disease.
• neuroorestoration is meant compensation of existing alterations and stimulation of structural and functional restoring of the injured nervous activity.
• patient refers to a warm-blooded animal, more preferably a human, who/which is awaiting or receiving medical care or is or will be the object of a medical procedure.
• human refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.
• the terms “treat”, “treating” and “treatment”, as used herein, are meant to include alleviating or abrogating a condition or disease and/or its attendant symptoms.
• prevent refers to a method of delaying or precluding the onset of a condition or disease and/or its attendant symptoms, barring a patient from acquiring a condition or disease, or reducing a patient's risk of acquiring a condition or disease.
• terapéuticaally effective amount means the amount of platelet lysate fraction of the invention which is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered.
• administration or a variant thereof (e.g., “administering"), means providing the platelet lysate fraction of the invention, alone or as part of a pharmaceutically acceptable solution, to the patient in whom/which the condition, symptom, or disorder is to be treated or prevented.
• Figure 1 Time course of treatment with platelet lysate and platelet lysate fractions.
• Platelet lysate (control) and fractions were added lh before erastin (A) or lh, 3h, 6h and 8h after erastin (B).
• Figure 3 Neuroprotective effect of 50k Da, II -50k Da, 30k Da, II-30kDa, 10k Da, and II- 10k Da fractions measured by resazurin.
• Treatment l h before erastin (E). The viability was measured and normalized to the control (non treated cells) n l.
• Figure 5 Neuroprotective effect of 3kDa and II-3kDa fractions measured by resazurin. Treatment of LUHMES cells lh, 3h, 6h or 8h after Erastin (E).
• Figure 6 Neuroprotective effect measured by cytometric assay of II -3k Da fraction.
• Figure 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 (Sodl*G86R)
• Veh Vehicle.
• Figure 8 Survival curve of male and female mice treated by vehicle and H-PPL diluted preparation.
• Male Tg male FVB-Tg (Sodl*G86R)
• Female Tg Female FVB-Tg (Sodl*G86R).
• Figure 9A Body weight evolution of male mice treated three times a week by vehicle and H-3kDa preparation.
• Male WT Male wild-type
• Male Tg male FVB-Tg (Sodl*G86R)
• Veh Vehicle.
• Figure 9B Body weight evolution of female mice treated three times a week by vehicle and H-3kDa preparation.
• Female WT Female wild-type
• Female Tg Female FVB-Tg (Sodl*G86R)
• Veh Vehicle.
• Figure 10 Survival curve of male and female mice treated three times a week by vehicle and H-3kDa preparation.
• Male Tg male FVB-Tg (Sodl*G86R)
• Female Tg Female FVB-Tg (Sodl*G86R).
• Figure 11A Body weight evolution of male mice treated six times a week by vehicle and H-3kDa preparation.
• Male WT Male wild-type
• Male Tg male FVB-Tg (Sodl*G86R)
• Veh Vehicle.
• Figure 11B Body weight evolution of female mice treated six times a week by vehicle and H-3kDa preparation.
• Female WT Female wild-type
• Female Tg Female FVB-Tg (Sodl*G86R), Veh:Vehicle.
• Figure 12 Survival curve of male and female mice treated six times a week by vehicle and H-3kDa preparation.
• Male Tg male FVB-Tg (Sodl*G86R)
• Female Tg Female FVB-Tg (Sodl*G86R).
• H-lOkDa fraction heat-treated platelet lysate 10 kDa fraction
• H-30kDa fraction heat-treated platelet lysate 30 kDa fraction
• H-3kDa fraction heat-treated platelet lysate 3 kDa fraction
• H-50kDa fraction heat-treated platelet lysate 50 kDa fraction
• H-pHPL heat-treated pooled human platelet lysate
• H-pHPL-GB pooled human platelet lysate mixed with glass beads (GB) and subjected to heat-treament
• HPL human platelet lysate
• H-PPL heat-treated platelet pellet lysate
• ICV intra cerebro-ventricular
• PAS platelet additive solution
• PBS phosphate buffer saline
• pHPL pooled human platelet lysate
• PPL platelet pellet lysate
• PPL platelet pellet lysate from non-expired PC
• RBC red blood cells
• Platelet lysates were obtained from platelet concentrates (Etablatorium Frangais dii Sang. Lille, France). After centrifugation at 4600 x g for 20 minutes at room temperature, platelet pellets were washed twice and resuspended in PBS in 1/10 th of the initial volume. Then, platelet pellets were frozen (nitrogen) and thawed (37°C) three times, and centrifuged at 4600 x g for 20 minutes at room temperature.
• PPL Platinum Pellet Lysate
• H- PPL Heat-treated Platelet Pellet Lysate
• 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-50kDa fraction, H-30kDa fraction, I I- 10k Da fraction and H-3kDa fraction when obtained from H-PPL.
• LUHMES cells were obtained from Dr. Scholz's laboratory (University of Konstanz, Germany) and cultured as described 10
• undifferentiated LUHMES cells were propagated using NunclonTM (Nunc, Roskilde, Denmark) plastic cell culture flasks and multi-well plates that were pre-coated with 50 ⁇ g/mL poly-L-ornithine and 1 ⁇ g/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA) in water for 3h at 37°C. After removal of the coating solution, culture flasks were washed with sterile distilled water and air-dried.
• the proliferation medium was Advanced Dulbecco' s modified Eagle's medium (Advanced DMEM)/F12 containing lx N-2 supplement (Invitrogen, Düsseldorf, Germany), 2 mM L- glutamine (Gibco, Rockville, MD, USA) and 40 ng/mL recombinant bFGF (R&D Systems).
• Advanced DMEM Advanced Dulbecco' s modified Eagle's medium
• F12 containing lx N-2 supplement Invitrogen, Düsseldorf, Germany
• 2 mM L- glutamine Gibco, Rockville, MD, USA
• 40 ng/mL recombinant bFGF R&D Systems
• 2xl0 6 LUHMES were seeded and grown into a T75 flask in proliferation medium for 48 h, then in Advanced DMEM/F12 containing lx N-2 supplement, 2 mM L-glutamine (Gibco), 1 mM dibutyryl cAMP (Sigma-Aldrich), 1 ⁇ g/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.
• the viability of LHUMES cells was evaluated by cytometry assay in 24 wells by using propidium iodide incorporation ( Figure 2) and compared to the control or to the different platelet lysates.
• the cyto meter used for the experiments is the CyAnTM model with a 488 nm laser (Beckman Coulter).
• the viability was also measured by a resazurin assay performed in 96 wells at 7 days of differentiation and 24h after treatments with 5 Ok Da, H-50kDa, 30kDa, H-30kDa, 10k Da, and H- I OkDa fractions.
• the assay is performed on the cell culture without trysinitation ( Figure 3).
• the platelet lysate H-3kDa 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 ( Figure 4).
• Akt inhibitor was performed and the inhibitor MK-2206 was added to the medium at 5 ⁇ lh before exposure to platelet lysate fractions.
• the treatment with platelet lysate H-3kDa fraction was performed lh before exposure to Erastin andlh, 3h, 6h, and 8h after exposure to Erastin.
• 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 ⁇ g/ ⁇ L. 6.
• Results are expressed as the mean + standard error of the mean (SEM).
• SEM standard error of the mean
• the platelet lysate fractions protect LUHMES cells from death induced by Erastin and display strong neuroprotective effect.
• 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.
• 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 x g, 4°C) to obtain the so-called HT-pHPL.
• An another part of the pHPL was mixed with 0,5 g/mL of glass beads (BEAD-002-lkg of 2 mm of diameter, from Labbox) and CaCl 2 (23mM final concentration; C4901 Calcium chloride anhydrous powder, from Sigma-Aldrich) under stirring for lh.
• H-pHPL-GB The heat-treated platelet lysate 3k Da 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).
• H-pHPL-GB 500 ⁇ L of H-pHPL-GB is added to the filter device inserted into the collect tube. and centrifuged at 14000 x g for 30 minutes at 4°C with a fixed angle rotor at 40°.
• the filtrate or the heat-treated platelet lysate fraction which is the lower part lower than the cutoff, is called H-3kDa because it was obtained from a heat- treated platelet lysate.
• the platelet lysate fractions were then aliquoted and stored at -80°C for further experiments.
• LUHMES cells were obtained and prepared as described in example 1.
• H-3kDa 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-3kDa fraction was added into the medium lh before treatment with Erastin (E).
• 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 CyAnTM model with a 488 nm laser (Beckman Coulter).
• 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 ⁇ g/ ⁇ L.
• the H-3kDa fraction protects LUHMES cells from death by ferroptosis and display strong neuroprotective effect.
• This example exhibits the potential of the heat-treated platelet lysate 3kDa 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-3kDa fraction is more suitable and more efficient for use in biotherapy, especially through brain administration.
• mice enrolled were FVB-Tg (Sodl*G86R) MlJwg/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 perfomed by qPCR (from tail biopsy). Animal are identified with earrings. ⁇ . Experimental protocol
• 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.
• body weight and neuroscore i.e. body weight and neuroscore
• Preparation 1 H-PPL as described in example 1, section 1 and diluted at 50 % in PBS (H- PPL diluted).
• 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 (D 102-D 112).
• H-3kDa 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.
• H-3kDa fraction had no effect in male and female body weight.
• body weight loss initiation (compare to Tg mice with vehicle) but, H-3kDa 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.
• H-3kDa has an excellent safety profile and a great efficacy with a classical sex-dose related effect.
• treatment with H-3kDa 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 3kDa fraction according to the invention in order to induce neuroprotective ability.

Landscapes

• Health & Medical Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Hematology (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Neurosurgery (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Neurology (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Cell Biology (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Developmental Biology & Embryology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Hospice & Palliative Care (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Psychiatry (AREA)
• Virology (AREA)
• Wood Science & Technology (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (9)

Applications Claiming Priority (2)

Publications (1)

Family

ID=60083230

Family Applications (1)

Country Status (11)

Cited By (2)

Families Citing this family (2)

Citations (1)

Family Cites Families (5)

• 2018
  • 2018-09-26 TW TW107133811A patent/TW201914595A/zh unknown
  • 2018-09-27 CA CA3076858A patent/CA3076858A1/en active Pending
  • 2018-09-27 JP JP2020517424A patent/JP2020535163A/ja active Pending
  • 2018-09-27 US US16/650,070 patent/US20230135837A1/en not_active Abandoned
  • 2018-09-27 EP EP18782679.7A patent/EP3687554A1/en not_active Withdrawn
  • 2018-09-27 RU RU2020112898A patent/RU2020112898A/ru unknown
  • 2018-09-27 BR BR112020006316-9A patent/BR112020006316A2/pt not_active IP Right Cessation
  • 2018-09-27 AU AU2018343875A patent/AU2018343875A1/en not_active Abandoned
  • 2018-09-27 WO PCT/EP2018/076244 patent/WO2019063683A1/en not_active Ceased
  • 2018-09-27 CN CN201880061493.6A patent/CN111107857A/zh active Pending
• 2020
  • 2020-03-22 IL IL273482A patent/IL273482A/en unknown

Patent Citations (1)

Non-Patent Citations (16)

Also Published As

Similar Documents

Legal Events