WO2020243808A1 - Method for the preparation and prolonged storage of growth factors and cytokines obtained from platelet rich plasma - Google Patents

Method for the preparation and prolonged storage of growth factors and cytokines obtained from platelet rich plasma Download PDF

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Publication number
WO2020243808A1
WO2020243808A1 PCT/CA2019/000083 CA2019000083W WO2020243808A1 WO 2020243808 A1 WO2020243808 A1 WO 2020243808A1 CA 2019000083 W CA2019000083 W CA 2019000083W WO 2020243808 A1 WO2020243808 A1 WO 2020243808A1
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Prior art keywords
rich plasma
platelet rich
plasma component
composition
months
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PCT/CA2019/000083
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French (fr)
Inventor
Anthony Galea
Irina Brokhman
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Antnor Limited
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Priority to PCT/CA2019/000083 priority Critical patent/WO2020243808A1/en
Publication of WO2020243808A1 publication Critical patent/WO2020243808A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2066—IL-10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue

Definitions

  • the application is directed generally to medicine, and more particularly to methods for the long term storage of viable compositions useful, in among other things, the treatment of damaged and/or injured connective tissues including chronic tendinosis, chronic muscle tears (tendinitis), cartilage tears, chronic degenerative joint conditions such as osteoarthritis as well as chronic inflammatory skin diseases including, atopic dermatitis and chronic wounds.
  • viable compositions may be used in dental treatments including dental implants, and for cosmetic applications.
  • Platelet Rich Plasma (PRP) therapy is employed for the treatment of damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a mammal.
  • Platelet Rich Plasma is blood plasma that has been enriched with platelets, typically by centrifugation of blood to isolate a platelet rich component.
  • Platelets contain proteins including growth factors and cytokines that are effective for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, and skin inflammatory disorders. These proteins are useful in the treatment of periodontal bone inflammation which has applications for dental implant procedures given the inflammation caused by dental implant hardware. In addition, these proteins are also effective for cosmetic purposes.
  • These cytokines and growth factors include IL-4,10,13, VEGF, PDGF, TGFp, FGF, IGF and IGF.
  • the platelets In order for the growth factors and the cytokines to be released from the platelets, the platelets must be activated. PRP fractions are typically collected in the presence of a blood anticoagulant because coagulation of the platelets leads to premature activation of the platelets resulting in the growth factors and the cytokines being released from the platelets prematurely. Some PRP preparation processes take up to thirty minutes. Where no anticoagulant is employed in such processes, the blood may clot prior to the isolation of the PRP component.
  • activation of the platelets can be achieved through a number of means. These means include addition of CaCl, addition of thrombin, freezing and thawing of platelets and ultrasonic waves.
  • compositions containing growth factors and cytokines obtained from activated platelets can typically only be stored for about six hours at room temperature before losing biological activity.
  • a typical PRP treatment protocol requires several follow up injections over a period of three to nine months.
  • Typical PRP compositions are not stored between treatments. It is therefore necessary to draw blood from the patient on each visit and often expensive kits are employed for each PRP treatment. This regimen is both invasive and costly.
  • compositions comprising growth factors and cytokines obtained from platelets that are effective for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders, cosmetic applications and periodontal bone inflammation, and which are capable of being stored for up to at least eighteen months without diminishing the biological activity of the composition.
  • Described is a method for producing a composition comprising proteins including cytokines and growth factors obtained from platelet rich plasma.
  • the cytokines and growth factors in the composition retain biological activity and therapeutic effectiveness after prolonged storage for up to at least eighteen months when frozen.
  • the composition is can be stored at between -50°C and -196°C but is preferably stored at between -50°C and -80°C.
  • the composition can be stored at these temperatures by employing an ultra-low freezer. Storage at - 196°C can be accomplished by methods known in the art including using liquid nitrogen.
  • the composition After such prolonged storage of a PRP fraction of the composition in a frozen state, the composition remains effective after thawing the PRP fraction for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation.
  • the composition comprises IL-4,10,13, VEGF, PDGF, IGF, FGF, TGFp. Also described is the composition produced by the method described herein.
  • an autologous composition comprising regenerative growth factors and cytokines obtained from platelets, the method comprising the following steps:
  • the platelet rich plasma component passes through a small pore filter to produce the autologous composition comprising regenerative growth factors and cytokines derived from platelets; and collecting the autologous composition.
  • an autologous composition comprising regenerative growth factors and cytokines obtained from platelets produced by the method described herein.
  • storage stable autologous composition comprising regenerative growth factors and cytokines derived from platelets, the composition comprising biologically active concentrations of IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1, FGF and VEGF, wherein the autologous composition retains regenerative biological activity when the platelet rich plasma component is frozen and stored for up to at least eighteen months.
  • Figure 1 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the thrombin and mechanically activated PRP.
  • Figure 2 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the thrombin and mechanically activated PRP.
  • Figure 3 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the thrombin and mechanically activated PRP.
  • Figure 4 is a plot of PDGF concentration in pg/ml showing a comparison of the levels of PDGF in the thrombin and mechanically activated PRP.
  • Figure 5 is a plot of VEGF concentration in pg/ml showing a comparison of the levels of VEGF in the thrombin and mechanically activated PRP.
  • Figure 6 is a plot of TGFp concentration in pg/ml showing a comparison of the levels of TGF in the thrombin and mechanically activated PRP.
  • Figure 7 is a plot of IL-4 concentration in pg/ml showing a comparison of the levels of IL-4 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.
  • Figure 8 is a plot of lL-10 concentration in pg/ml showing a comparison of the levels of IL-10 in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 9 is a graph showing a comparison between the levels of IL-13 in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 10 is a graph showing a comparison between the levels of PGDF in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 1 1 is a graph showing a comparison between the levels of VEGF in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
  • Figure 12A is a graph showing a comparison between the levels of TGFp in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
  • Figure 12B is a graph indicating instability of TGF-bI concentration upon different conditions.
  • Figure 13 is a graph showing a comparison between the levels of IGF1 in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
  • Figure 14 is a graph showing a comparison between the levels of hFGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 15 is a graph showing a comparison between the levels of IL-4 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 16 is a graph showing a comparison between the levels of IL-10 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 17 is a graph showing a comparison between the levels of IL-10 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 18 is a graph showing a comparison between the levels of PDGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 19 is a graph showing a comparison between the levels of VEGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
  • Figure 20a is a plot showing point values according to the Visual Analog Pain Scale (VAS) among seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
  • VAS Visual Analog Pain Scale
  • Figure 20b is a plot showing point values according to the WOMAC index for average levels of pain among seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
  • Figure 20c is a plot showing point values according to the WOMAC index for average levels of stiffness among the seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
  • Figure 20d is a plot showing point values according to the WOMAC index for average levels of daily activity capabilities among the seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up. DETAILED DESCRIPTION OF THE INVENTION
  • the disclosure relates to a method for producing a stable composition
  • a platelet rich plasma component wherein the composition retains biological activity and therapeutic efficacy after prolonged storage for up to at least eighteen months when the platelet rich plasma component is frozen and stored preferably at between -50°C and -196°C for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation caused by for example dental implants.
  • the platelet rich plasma component of the composition preferably includes the following therapeutically active proteins: IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1 , FGF and VEGF.
  • the platelet rich plasma component contains a source of regenerative biological factors and anti-inflammatory cytokines and growth factors.
  • the composition also provides a powerful and cost effective treatment of degenerative conditions including osteoarthritis, chronic tendinosis and chronic muscle tears as well as skin inflammatory disorders and periodontal bone inflammation caused by dental procedures such as dental implants given the inflammation caused by dental implant hardware in such procedures.
  • the composition also provides powerful and cost effective cosmetic applications.
  • treatment includes regenerative and palliative treatment, wherein pain and/or inflammation is reduced in the subject.
  • the described method for producing and storing the stable composition of platelet rich plasma components preferably including regenerative growth factors and cytokines derived from platelets comprises the step of collecting a mammal’s autologous physiological fluid, preferably blood by an aseptic technique.
  • a mammal is a human.
  • the compositions and methods hereof are also suitable for a wide range of veterinary applications, for example for the treatment of horses, dogs and camels.
  • the site of venipuncture and the surface of the collection tubes may be cleaned with a 2 percent tincture of iodine solution. Before any cleansing of the site is begun, the patient may be asked about any allergy to iodine.
  • the collection container covers are cleaned with 80% alcohol solution also to avoid possible contamination before blood collection.
  • the collection of blood is preferably carried out in the presence of an anticoagulant.
  • the anticoagulant is 4% citric acid.
  • the ratio is 9.5 parts of whole blood (9.5cc) : 0.5 (0.5cc) of 4% citric acid.
  • the blood is then subjected to centrifugation preferably for about 30 sec, at about 7500 rpm to isolate the PRP fraction.
  • centrifugation preferably for about 30 sec, at about 7500 rpm to isolate the PRP fraction.
  • the blood can be processed or the PRP fraction can be isolated using commercially available kits such as HarvestTM and ArthrexTM.
  • the next step involves collecting the PRP fraction and dividing it into aliquots for future processing using a sterile technique or for storage.
  • the procedure is carried out in a sterile environment (laminar flow hood with HEPA filters).
  • Two to four cc of the storage stable autologous composition containing biologically active agents are carefully drawn by sterile syringe and needle.
  • Prolonged storage of PRP ingredients containing product is accomplished by freezing the aliquots.
  • the aliquots are frozen at a temperature in the range of about -50 °C to about -196 °C and most preferably at about -80°C followed by storing for up to eighteen months or longer while maintaining acceptable biological activity and therapeutic efficacy of the autologous composition.
  • the PRP fraction of the autologous composition can be frozen and stored while maintaining acceptable biological activity and therapeutic efficacy for at least eighteen months.
  • the aliquots of frozen PRP are then thawed and preferably brought to room temperature.
  • the thawed PRP fraction is activated mechanically by passing the PRP fraction through a small pore size filter and collected in a sterile plastic vial.
  • the pore size of the filter is in the range of about 0.15pm to about l pm. Most preferably the pore size of the filter is about 0.22pm.
  • An example of a filter that is suitable for carrying out this method is Millipore Corporation, Millex-GP 33mm PES .22pm Sterile, Catalog N SLGPM33RS.
  • the isolated PRP fraction is preferably passed through the filter by adding the PRP fraction to a syringe and delivering the PRP fraction through the filter through the action of the syringe.
  • the platelet rich plasma component through the small pore filter mechanically activates the platelets in the PRP fraction. This releases the cytokines and growth factors from the platelets. Platelet cellular debris is caught in the filter.
  • the liquid composition that is obtained after the PRP fraction passes through the filter is acellular and comprises regenerative growth factors and cytokines derived from platelets.
  • the acellular autologous composition that is then obtained is substantially free of cellular debris as a result of the filtration. It is beneficial to avoid the introduction of cellular debris in a joint or tissue into which the autologous composition is injected after the cytokines from the platelets are released. This cellular debris would eventually need to be removed from the injection site which results in an energy cost and potentially could adversely affect the outcomes of the treatment.
  • the autologous composition is then passed a second time through a separate small pore filter having a pore size in the range of about 0.15pm to about l pm, and preferably about 0.22pm. It is possible to employ the same filter as for the first pass through of the PRP fraction. However, this is not preferred due to potential filter occlusion.
  • the method of PRP mechanical activation of the present disclosure leads to the same cytokines and growth factors as a thrombin activation.
  • the PRP fraction can be stored in frozen state for at least eighteen months without decreasing its biological activity and reducing IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1, FGF and VEGF concentrations.
  • the autologous composition may then be administered to a patient for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation caused by for example dental implants.
  • the properties of the autologous composition comprising regenerative growth factors and cytokines obtained from platelets are further described with the aid of the following illustrative examples.
  • Example 1 Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets to Levels of Cytokines and Growth Factors Obtained Through Activation of Platelets with Bovine Thrombin
  • Blood was obtained from patients and a PRP fraction of the blood was obtained from each patient according to the method of the present disclosure.
  • the platelets in the PRP fraction were activated according to the following procedure: Bovine thrombin was added: 100 units for l cc PRP. Samples were incubated at room temperature for 10 min to allow plasma to clot.
  • the platelets in the PRP fraction were activated mechanically according to the procedure set out in the present disclosure by passing the PRP fraction through a filter having a pore size of about 0.22pm.
  • the PRP fraction was passed through a filter a second time.
  • Figure 1 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL-4 in the composition where the platelets were activated by thrombin compared to the level of IL-4 in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Figure 2 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL-10 in the composition where the platelets were activated by thrombin compared to the level of IL-10 in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Figure 3 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL- 13 in the composition where the platelets were activated by thrombin compared to the level of IL-13 in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Figure 4 is a plot of PDGF concentration in pg/ml showing a comparison of the level of PDGF in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of PDGF in the composition where the platelets were activated by thrombin compared to the level of PDGF in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Figure 5 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the composition obtained from platelets activated by thrombin and platelets activated from the VEGF fraction. The results show that there is no statistically significant difference in the level of VEGF in the composition where the platelets were activated by thrombin compared to the level of VEGF in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Figure 6 is a plot of TGFp concentration in pg/ml showing a comparison of the level of TGFp in the composition obtained from platelets activated by thrombin and platelets activated from the TGFp fraction. The results show that there is no statistically significant difference in the level of TGFp in the composition where the platelets were activated by thrombin compared to the level of TGFp in the composition where the platelets were activated mechanically according to the method of the present disclosure.
  • Example 2 Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets in Fresh Autologous Composition and in Composition Stored for 9 Months at -80°C
  • the autologous composition was obtained according to the method of the present disclosure with a variation to the method in that the isolated PRP fraction was activated mechanically by passing the PRP fraction through a small pore size filter of pore size in the range of about 0.15pm to about 1 pm and collected in a sterile plastic vial prior to freezing and storage The composition was then divided into aliquots of 2-4cc by carefully drawing of the composition by a sterile syringe and needle. The aliquots were then frozen and stored at -80°C for 9 months.
  • Figure 7 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the fresh autologous composition to the level of IL-4 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-4 in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 8 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the fresh autologous composition to the level of IL-10 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-10 in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 9 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the fresh autologous composition to the level of IL-13 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-13 in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 10 is a plot of PGDF concentration in pg/ml showing a comparison of the level of PGDF in the fresh autologous composition to the level of PGDF in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of PGDF in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 1 1 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the fresh autologous composition to the level of VEGF in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of VEGF in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 12 A is a plot of TGFp- 1 concentration in pg/ml showing a comparison of the level of TGFP in the fresh autologous composition to the level of TGFP- 1 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of TGFp-1 in the autologous composition is preserved after storage for nine months at -80°C.
  • Figure 12 B is a plot of TGFp-1 concentration in pg/ml showing a comparison of the level of TGFp in the fresh and lyophilized autologous blood product (a combination of incubated blood and PRP). The results show that a lyophilizing method leads to significant decreasing of the level of TGFp- 1 whereas the freezing method (A) surprisingly preserves normal concentration.
  • Figure 13 is a plot of IGF 1 concentration in pg/ml showing a comparison of the level of IGF 1 in the fresh autologous composition to the level of IGF 1 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IGF1 in the autologous composition is preserved after storage for nine months at -80°C.
  • Example 3- Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets in Fresh Autologous Composition and in Composition Stored for 18 Months at -50 to -80°C
  • the autologous composition was obtained according to the method of the present disclosure with a variation to the method in that the isolated PRP fraction was activated mechanically by passing the PRP fraction through a small pore size filter of pore size in the range of about 0.15pm to about 1 pm and collected in a sterile plastic vial prior to freezing and storage The composition was then divided into aliquots of 2-4cc by carefully drawing of the composition by a sterile syringe and needle. The aliquots were then frozen and stored at between -50°C and -80°C for 18 months.
  • Figure 14 is a plot of hFGF (human fibroblast growth factor) concentration in pg/ml showing a comparison of the level of hFGF in the fresh autologous composition to the level of hFGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of hFGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • hFGF human fibroblast growth factor
  • Figure 15 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the fresh autologous composition to the level of IL-4 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-4 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • Figure 16 is a plot of IL- 10 concentration in pg/ml showing a comparison of the level of IL-10 in the fresh autologous composition to the level of IL- 10 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-10 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • Figure 17 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the fresh autologous composition to the level of IL-13 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL- 13 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • Figure 18 is a plot of PDGF concentration in pg/ml showing a comparison of the level of PDGF in the fresh autologous composition to the level of PDGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of PDGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • Figure 19 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the fresh autologous composition to the level of VEGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of VEGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
  • the scheduled treatment regimen was one dose per week for four weeks as an intraarticular (IA) injection into the affected knee. Subjects were followed up after 29 days and 57 days with planned follow up for up to six months. Eight participants in the frozen PRP arm and nine participants in the PRP arm were enrolled.
  • IA intraarticular
  • results obtained from two follow ups confirm no superiority of conventional PRP over frozen PRP treatments.
  • results obtained for the patients in terms of pain (Visual Analog Pain Scale (VAS) and Womac pain index, stiffness experienced (Womac stiffness scale) and average levels of daily activity capabilities (Womac DA).
  • VAS Visual Analog Pain Scale
  • Womac stiffness scale stiffness experienced
  • Womac DA average levels of daily activity capabilities

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Abstract

Described is a method of producing an autologous composition comprising regenerative growth factors and cytokines obtained from platelets. The method comprises the steps of collecting or providing blood from a mammal; delivering the blood to a receptacle; centrifuging the blood to separate a platelet rich plasma component from the blood; collecting the platelet rich plasma component; freezing and storing the platelet rich plasma component; thawing the platelet rich plasma component and passing the platelet rich plasma component through a small pore filter to provide the activated autologous composition. The storage stable autologous composition retains regenerative biological activity for up to at least eighteen months after storage of the platelet rich plasma component in a frozen state. Also described is a storage stable autologous composition comprising regenerative growth factors and cytokines obtained from platelets produced by the method as described.

Description

TITLE
METHOD FOR THE PREPARATION AND PROLONGED STORAGE OF GROWTH FACTORS AND CYTOKINES OBTAINED FROM PLATELET RICH PLASMA
TECHNICAL FIELD
The application is directed generally to medicine, and more particularly to methods for the long term storage of viable compositions useful, in among other things, the treatment of damaged and/or injured connective tissues including chronic tendinosis, chronic muscle tears (tendinitis), cartilage tears, chronic degenerative joint conditions such as osteoarthritis as well as chronic inflammatory skin diseases including, atopic dermatitis and chronic wounds. In addition, the viable compositions may be used in dental treatments including dental implants, and for cosmetic applications.
BACKGROUND
Platelet Rich Plasma (PRP) therapy is employed for the treatment of damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions and skin inflammatory disorders in a mammal. Platelet Rich Plasma is blood plasma that has been enriched with platelets, typically by centrifugation of blood to isolate a platelet rich component. Platelets contain proteins including growth factors and cytokines that are effective for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, and skin inflammatory disorders. These proteins are useful in the treatment of periodontal bone inflammation which has applications for dental implant procedures given the inflammation caused by dental implant hardware. In addition, these proteins are also effective for cosmetic purposes. These cytokines and growth factors include IL-4,10,13, VEGF, PDGF, TGFp, FGF, IGF and IGF.
In order for the growth factors and the cytokines to be released from the platelets, the platelets must be activated. PRP fractions are typically collected in the presence of a blood anticoagulant because coagulation of the platelets leads to premature activation of the platelets resulting in the growth factors and the cytokines being released from the platelets prematurely. Some PRP preparation processes take up to thirty minutes. Where no anticoagulant is employed in such processes, the blood may clot prior to the isolation of the PRP component.
For PRP fractions that are collected in the presence of an anticoagulant such as sodium citrate, activation of the platelets can be achieved through a number of means. These means include addition of CaCl, addition of thrombin, freezing and thawing of platelets and ultrasonic waves.
Each of these activation methods has drawbacks. Repeated freeze-thaw cycles lead to degradation of biologically active proteins such as TGF and PDGF. Thrombin animal bovine protein can be a source of pathogens or lead to allergic reaction. Human thrombin employed in certain PRP kits is very expensive. Addition of CaCl interferes with the ability to make important measurements of cytokine and growth factor levels and provides a source of impurities into PRP compositions. Lastly, there are regulatory issues with the use of ultrasonic waves and this technique has not been demonstrated to have reliable effectiveness.
There is therefore a need for a more efficient manner of activating platelets thereby releasing the growth factors and cytokines contained therein.
Compositions containing growth factors and cytokines obtained from activated platelets can typically only be stored for about six hours at room temperature before losing biological activity. A typical PRP treatment protocol requires several follow up injections over a period of three to nine months. Typical PRP compositions are not stored between treatments. It is therefore necessary to draw blood from the patient on each visit and often expensive kits are employed for each PRP treatment. This regimen is both invasive and costly.
There is therefore a need for a method of producing a composition comprising growth factors and cytokines obtained from platelets that are effective for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders, cosmetic applications and periodontal bone inflammation, and which are capable of being stored for up to at least eighteen months without diminishing the biological activity of the composition.
SUMMARY OF THE DISCLOSURE
Described is a method for producing a composition comprising proteins including cytokines and growth factors obtained from platelet rich plasma. The cytokines and growth factors in the composition retain biological activity and therapeutic effectiveness after prolonged storage for up to at least eighteen months when frozen. The composition is can be stored at between -50°C and -196°C but is preferably stored at between -50°C and -80°C. The composition can be stored at these temperatures by employing an ultra-low freezer. Storage at - 196°C can be accomplished by methods known in the art including using liquid nitrogen. After such prolonged storage of a PRP fraction of the composition in a frozen state, the composition remains effective after thawing the PRP fraction for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation. Preferably, the composition comprises IL-4,10,13, VEGF, PDGF, IGF, FGF, TGFp. Also described is the composition produced by the method described herein.
According to one aspect, there is provided a method of producing an autologous composition comprising regenerative growth factors and cytokines obtained from platelets, the method comprising the following steps:
providing blood from a mammal;
delivering the blood to a receptacle;
centrifuging the blood to separate a platelet rich plasma component from the blood;
collecting the platelet rich plasma component;
freezing the platelet rich plasma component;
storing the platelet rich plasma component;
thawing the platelet rich plasma component;
passing the platelet rich plasma component through a small pore filter to produce the autologous composition comprising regenerative growth factors and cytokines derived from platelets; and collecting the autologous composition.
According to another aspect, there is provided an autologous composition comprising regenerative growth factors and cytokines obtained from platelets produced by the method described herein.
According to another aspect, there is provided storage stable autologous composition comprising regenerative growth factors and cytokines derived from platelets, the composition comprising biologically active concentrations of IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1, FGF and VEGF, wherein the autologous composition retains regenerative biological activity when the platelet rich plasma component is frozen and stored for up to at least eighteen months.
DESCRIPTION OF THE DRAWINGS
Figure 1 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the thrombin and mechanically activated PRP.
Figure 2 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the thrombin and mechanically activated PRP.
Figure 3 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the thrombin and mechanically activated PRP.
Figure 4 is a plot of PDGF concentration in pg/ml showing a comparison of the levels of PDGF in the thrombin and mechanically activated PRP.
Figure 5 is a plot of VEGF concentration in pg/ml showing a comparison of the levels of VEGF in the thrombin and mechanically activated PRP. Figure 6 is a plot of TGFp concentration in pg/ml showing a comparison of the levels of TGF in the thrombin and mechanically activated PRP.
Figure 7 is a plot of IL-4 concentration in pg/ml showing a comparison of the levels of IL-4 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.
Figure 8 is a plot of lL-10 concentration in pg/ml showing a comparison of the levels of IL-10 in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 9 is a graph showing a comparison between the levels of IL-13 in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 10 is a graph showing a comparison between the levels of PGDF in fresh and 9 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 1 1 is a graph showing a comparison between the levels of VEGF in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
Figure 12A is a graph showing a comparison between the levels of TGFp in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
Figure 12B is a graph indicating instability of TGF-bI concentration upon different conditions.
Figure 13 is a graph showing a comparison between the levels of IGF1 in fresh and 9 months stored stable solutions of Platelet Rich Plasma Ingredients.
Figure 14 is a graph showing a comparison between the levels of hFGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 15 is a graph showing a comparison between the levels of IL-4 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients. Figure 16 is a graph showing a comparison between the levels of IL-10 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 17 is a graph showing a comparison between the levels of IL-10 in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 18 is a graph showing a comparison between the levels of PDGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 19 is a graph showing a comparison between the levels of VEGF in fresh and 18 months stored solutions of Platelet Rich Plasma Ingredients.
Figure 20a is a plot showing point values according to the Visual Analog Pain Scale (VAS) among seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
Figure 20b is a plot showing point values according to the WOMAC index for average levels of pain among seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
Figure 20c is a plot showing point values according to the WOMAC index for average levels of stiffness among the seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up.
Figure 20d is a plot showing point values according to the WOMAC index for average levels of daily activity capabilities among the seventeen patients tested with conventional PRP and previously frozen PRP. Values are provided for a baseline, 29 day follow up and 57 day follow up. DETAILED DESCRIPTION OF THE INVENTION
The disclosure relates to a method for producing a stable composition comprising a platelet rich plasma component wherein the composition retains biological activity and therapeutic efficacy after prolonged storage for up to at least eighteen months when the platelet rich plasma component is frozen and stored preferably at between -50°C and -196°C for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation caused by for example dental implants.
The platelet rich plasma component of the composition preferably includes the following therapeutically active proteins: IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1 , FGF and VEGF.
IL-4,10, 13, PDGF, IGF1 , TGF- b, FGF are contained in the platelet a-granules and are delivered to the composition by the platelet rich plasma component. The ability to store the composition for a prolonged period of time provides a potent and accessible bioactive autologous product that is efficient and cost effective to administer to patients. Thus, the storage stable composition of therapeutically active proteins included in the platelet rich plasma component provides a source of regenerative biological factors and anti-inflammatory cytokines and growth factors. The composition also provides a powerful and cost effective treatment of degenerative conditions including osteoarthritis, chronic tendinosis and chronic muscle tears as well as skin inflammatory disorders and periodontal bone inflammation caused by dental procedures such as dental implants given the inflammation caused by dental implant hardware in such procedures. In addition, the composition also provides powerful and cost effective cosmetic applications.
As used herein,“treatment” includes regenerative and palliative treatment, wherein pain and/or inflammation is reduced in the subject.
The described method for producing and storing the stable composition of platelet rich plasma components preferably including regenerative growth factors and cytokines derived from platelets comprises the step of collecting a mammal’s autologous physiological fluid, preferably blood by an aseptic technique. Preferably, the mammal is a human. However the compositions and methods hereof are also suitable for a wide range of veterinary applications, for example for the treatment of horses, dogs and camels.
The site of venipuncture and the surface of the collection tubes may be cleaned with a 2 percent tincture of iodine solution. Before any cleansing of the site is begun, the patient may be asked about any allergy to iodine. The collection container covers are cleaned with 80% alcohol solution also to avoid possible contamination before blood collection.
The collection of blood is preferably carried out in the presence of an anticoagulant. Preferably, the anticoagulant is 4% citric acid. Preferably, the ratio is 9.5 parts of whole blood (9.5cc) : 0.5 (0.5cc) of 4% citric acid. The blood is then subjected to centrifugation preferably for about 30 sec, at about 7500 rpm to isolate the PRP fraction. However, a person skilled the art will appreciate that the blood can be processed or the PRP fraction can be isolated using commercially available kits such as Harvest™ and Arthrex™.
The next step involves collecting the PRP fraction and dividing it into aliquots for future processing using a sterile technique or for storage. The procedure is carried out in a sterile environment (laminar flow hood with HEPA filters). Two to four cc of the storage stable autologous composition containing biologically active agents are carefully drawn by sterile syringe and needle. Prolonged storage of PRP ingredients containing product is accomplished by freezing the aliquots. Preferably the aliquots are frozen at a temperature in the range of about -50 °C to about -196 °C and most preferably at about -80°C followed by storing for up to eighteen months or longer while maintaining acceptable biological activity and therapeutic efficacy of the autologous composition. The PRP fraction of the autologous composition can be frozen and stored while maintaining acceptable biological activity and therapeutic efficacy for at least eighteen months.
After freezing and storage, the aliquots of frozen PRP are then thawed and preferably brought to room temperature. The thawed PRP fraction is activated mechanically by passing the PRP fraction through a small pore size filter and collected in a sterile plastic vial. Preferably the pore size of the filter is in the range of about 0.15pm to about l pm. Most preferably the pore size of the filter is about 0.22pm. An example of a filter that is suitable for carrying out this method is Millipore Corporation, Millex-GP 33mm PES .22pm Sterile, Catalog N SLGPM33RS. The isolated PRP fraction is preferably passed through the filter by adding the PRP fraction to a syringe and delivering the PRP fraction through the filter through the action of the syringe.
Passing the platelet rich plasma component through the small pore filter mechanically activates the platelets in the PRP fraction. This releases the cytokines and growth factors from the platelets. Platelet cellular debris is caught in the filter. The liquid composition that is obtained after the PRP fraction passes through the filter is acellular and comprises regenerative growth factors and cytokines derived from platelets. The acellular autologous composition that is then obtained is substantially free of cellular debris as a result of the filtration. It is beneficial to avoid the introduction of cellular debris in a joint or tissue into which the autologous composition is injected after the cytokines from the platelets are released. This cellular debris would eventually need to be removed from the injection site which results in an energy cost and potentially could adversely affect the outcomes of the treatment.
Preferably, the autologous composition is then passed a second time through a separate small pore filter having a pore size in the range of about 0.15pm to about l pm, and preferably about 0.22pm. It is possible to employ the same filter as for the first pass through of the PRP fraction. However, this is not preferred due to potential filter occlusion.
The method of PRP mechanical activation of the present disclosure leads to the same cytokines and growth factors as a thrombin activation.
The PRP fraction can be stored in frozen state for at least eighteen months without decreasing its biological activity and reducing IL-4, IL-10, IL-13, PDGF, TGF-b, IGF1, FGF and VEGF concentrations.
The autologous composition may then be administered to a patient for treating damaged and/or injured connective tissues, chronic tendinosis, chronic muscle tears and/or chronic degenerative joint conditions such as osteoarthritis, skin inflammatory disorders and periodontal bone inflammation caused by for example dental implants.
The properties of the autologous composition comprising regenerative growth factors and cytokines obtained from platelets are further described with the aid of the following illustrative examples.
Example 1 - Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets to Levels of Cytokines and Growth Factors Obtained Through Activation of Platelets with Bovine Thrombin
Blood was obtained from patients and a PRP fraction of the blood was obtained from each patient according to the method of the present disclosure.
For each patient, the platelets in the PRP fraction were activated according to the following procedure: Bovine thrombin was added: 100 units for l cc PRP. Samples were incubated at room temperature for 10 min to allow plasma to clot.
For each patient, the platelets in the PRP fraction were activated mechanically according to the procedure set out in the present disclosure by passing the PRP fraction through a filter having a pore size of about 0.22pm. The PRP fraction was passed through a filter a second time.
The data obtained is summarized in Figures 1-6.
Figure 1 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL-4 in the composition where the platelets were activated by thrombin compared to the level of IL-4 in the composition where the platelets were activated mechanically according to the method of the present disclosure. Figure 2 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL-10 in the composition where the platelets were activated by thrombin compared to the level of IL-10 in the composition where the platelets were activated mechanically according to the method of the present disclosure.
Figure 3 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of IL- 13 in the composition where the platelets were activated by thrombin compared to the level of IL-13 in the composition where the platelets were activated mechanically according to the method of the present disclosure.
Figure 4 is a plot of PDGF concentration in pg/ml showing a comparison of the level of PDGF in the composition obtained from platelets activated by thrombin and platelets activated from the PRP fraction. The results show that there is no statistically significant difference in the level of PDGF in the composition where the platelets were activated by thrombin compared to the level of PDGF in the composition where the platelets were activated mechanically according to the method of the present disclosure.
Figure 5 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the composition obtained from platelets activated by thrombin and platelets activated from the VEGF fraction. The results show that there is no statistically significant difference in the level of VEGF in the composition where the platelets were activated by thrombin compared to the level of VEGF in the composition where the platelets were activated mechanically according to the method of the present disclosure.
Figure 6 is a plot of TGFp concentration in pg/ml showing a comparison of the level of TGFp in the composition obtained from platelets activated by thrombin and platelets activated from the TGFp fraction. The results show that there is no statistically significant difference in the level of TGFp in the composition where the platelets were activated by thrombin compared to the level of TGFp in the composition where the platelets were activated mechanically according to the method of the present disclosure.
Example 2; - Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets in Fresh Autologous Composition and in Composition Stored for 9 Months at -80°C
The autologous composition was obtained according to the method of the present disclosure with a variation to the method in that the isolated PRP fraction was activated mechanically by passing the PRP fraction through a small pore size filter of pore size in the range of about 0.15pm to about 1 pm and collected in a sterile plastic vial prior to freezing and storage The composition was then divided into aliquots of 2-4cc by carefully drawing of the composition by a sterile syringe and needle. The aliquots were then frozen and stored at -80°C for 9 months.
Figure 7 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the fresh autologous composition to the level of IL-4 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-4 in the autologous composition is preserved after storage for nine months at -80°C.
Figure 8 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the fresh autologous composition to the level of IL-10 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-10 in the autologous composition is preserved after storage for nine months at -80°C.
Figure 9 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the fresh autologous composition to the level of IL-13 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-13 in the autologous composition is preserved after storage for nine months at -80°C.
Figure 10 is a plot of PGDF concentration in pg/ml showing a comparison of the level of PGDF in the fresh autologous composition to the level of PGDF in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of PGDF in the autologous composition is preserved after storage for nine months at -80°C.
Figure 1 1 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the fresh autologous composition to the level of VEGF in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of VEGF in the autologous composition is preserved after storage for nine months at -80°C.
Figure 12 A is a plot of TGFp- 1 concentration in pg/ml showing a comparison of the level of TGFP in the fresh autologous composition to the level of TGFP- 1 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of TGFp-1 in the autologous composition is preserved after storage for nine months at -80°C.
Figure 12 B is a plot of TGFp-1 concentration in pg/ml showing a comparison of the level of TGFp in the fresh and lyophilized autologous blood product (a combination of incubated blood and PRP). The results show that a lyophilizing method leads to significant decreasing of the level of TGFp- 1 whereas the freezing method (A) surprisingly preserves normal concentration.
Figure 13 is a plot of IGF 1 concentration in pg/ml showing a comparison of the level of IGF 1 in the fresh autologous composition to the level of IGF 1 in the autologous composition stored for nine months at -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IGF1 in the autologous composition is preserved after storage for nine months at -80°C. Example 3- Comparison of Levels of Cytokines and Growth Factors Obtained Through Mechanical Activation of Platelets in Fresh Autologous Composition and in Composition Stored for 18 Months at -50 to -80°C
The autologous composition was obtained according to the method of the present disclosure with a variation to the method in that the isolated PRP fraction was activated mechanically by passing the PRP fraction through a small pore size filter of pore size in the range of about 0.15pm to about 1 pm and collected in a sterile plastic vial prior to freezing and storage The composition was then divided into aliquots of 2-4cc by carefully drawing of the composition by a sterile syringe and needle. The aliquots were then frozen and stored at between -50°C and -80°C for 18 months.
Figure 14 is a plot of hFGF (human fibroblast growth factor) concentration in pg/ml showing a comparison of the level of hFGF in the fresh autologous composition to the level of hFGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of hFGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Figure 15 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the fresh autologous composition to the level of IL-4 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-4 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Figure 16 is a plot of IL- 10 concentration in pg/ml showing a comparison of the level of IL-10 in the fresh autologous composition to the level of IL- 10 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL-10 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Figure 17 is a plot of IL-13 concentration in pg/ml showing a comparison of the level of IL-13 in the fresh autologous composition to the level of IL-13 in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of IL- 13 in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Figure 18 is a plot of PDGF concentration in pg/ml showing a comparison of the level of PDGF in the fresh autologous composition to the level of PDGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of PDGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Figure 19 is a plot of VEGF concentration in pg/ml showing a comparison of the level of VEGF in the fresh autologous composition to the level of VEGF in the autologous composition stored for eighteen months at between -50°C and -80°C. The results show that there is no statistically significant difference in the respective levels and that accordingly the concentration of VEGF in the autologous composition is preserved after freezing for eighteen months at between -50°C and -80°C.
Example 4: Comparison of Effectiveness of Previously Frozen PRP to Conventional PRP
The effectiveness of previously frozen PRP was compared to conventional PRP through injections to 17 patients. This study was designed as a one-centre, active-controlled, randomized, double-blind, prospective study. Inclusion criteria were: age between 18-80 years, and clinical and radiographically confirmed evidence for knee osteoarthritis including VAS (Visual Analog Scale) >50. Exclusion criteria were: systemic metabolic diseases, psoriatic arthritis, pregnancy, malignancy, blood coagulation problems, systemic steroid and NIDS treatments, psychiatric disease, alcohol and gambling addictions. At the baseline visit (Day 1), subjects meeting eligibility requirements were randomized 1 : 1 to either a previously frozen PRP treatment arm or a PRP (control) treatment arm. The scheduled treatment regimen was one dose per week for four weeks as an intraarticular (IA) injection into the affected knee. Subjects were followed up after 29 days and 57 days with planned follow up for up to six months. Eight participants in the frozen PRP arm and nine participants in the PRP arm were enrolled.
As shown in figures 20a to 20d, results obtained from two follow ups (day 29 and Day 57) confirm no superiority of conventional PRP over frozen PRP treatments. According to data obtained for the patients in terms of pain (Visual Analog Pain Scale (VAS) and Womac pain index, stiffness experienced (Womac stiffness scale) and average levels of daily activity capabilities (Womac DA).
Although the invention has been described with reference to illustrative embodiments, it is to be understood that the invention is not limited to these precise embodiments. Numerous modifications, variations, and adaptations may be made to the particular embodiments of the invention described above without departing from the scope of the invention. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

Claims
1. A method of producing an autologous composition comprising regenerative growth factors and cytokines obtained from platelets, the method comprising the following steps:
providing blood from a mammal;
delivering the blood to a receptacle;
centrifuging the blood to separate a platelet rich plasma component from the blood; collecting the platelet rich plasma component;
freezing the platelet rich plasma component;
storing the platelet rich plasma component;
thawing the platelet rich plasma component;
passing the platelet rich plasma component through a small pore filter to produce the autologous composition comprising regenerative growth factors and cytokines derived from platelets; and
collecting the autologous composition.
2. The method of claim 1 wherein the platelet rich plasma component is frozen and stored at a temperature in the range of -50 °C to - 196 °C.
3. The method of claim 2 wherein the platelet rich plasma component is frozen and stored at -80°C.
4. The method of claim 1 wherein the platelet rich plasma component is stored for up to at least 18 months.
5. The method of claim 1 wherein the platelet rich plasma component is stored for between 9 months and 18 months.
6. The method of claim 2 wherein the platelet rich plasma component is stored for up to at least 18 months.
7. The method of claim 2 wherein the platelet rich plasma component is stored for between 9 months and at least 18 months.
8. The method of claim 1 wherein the pore size of the small pore filter is in the range of 0.15 pm to 1 pm.
9. The method of claim 8 wherein the autologous composition is delivered through a small pore filter having a pore size in the range of 0.15 pm to 1 pm.
10. The method of claim 1 wherein the pore size of the small pore filter is about 0.22 pm.
1 1. The method of claim 1 wherein the growth factors and cytokines include IL-4, IL-10, IL-
13, PDGF, TGF-b, IGF1 and VEGF.
12. The method according to claim 1 wherein the receptacle includes a quantity of an anticoagulant.
13. The method according to claim 12 wherein the anticoagulant is about 4% citric acid.
14. The method according to claim 1 wherein the receptacle is a tube.
15. The method of claim 1 further including the step of dividing the platelet rich plasma component into aliquots prior to freezing and storing the platelet rich plasma component.
16. The method of claim 15 wherein the aliquots are about 10 cc in volume of the platelet rich plasma component.
17. An autologous composition comprising regenerative growth factors and cytokines derived from platelets produced by the method of claim 1.
18. A storage stable autologous composition comprising regenerative growth factors and cytokines derived from platelets produced by the method of claim 12.
19. An autologous composition comprising regenerative growth factors and cytokines derived from platelets, the composition comprising biologically active concentrations of IL-4, IL-10, IL- 13, PDGF, TGF-b, IGF1 and VEGF, wherein the autologous composition retains regenerative biological activity for up to at least eighteen months when stored in a frozen state at a temperature in the range of -50 °C to -80 °C.
20. A method according to claim 1 wherein the autologous composition is administered to a patient.
PCT/CA2019/000083 2019-06-05 2019-06-05 Method for the preparation and prolonged storage of growth factors and cytokines obtained from platelet rich plasma WO2020243808A1 (en)

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