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 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 is 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 where the composition is capable of being stored for up to nine months without diminishing the biological activity of the composition.

SUMMARY OF THE DISCLOSURE

Described is a method for producing a stable 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 nine months when frozen. The composition is preferably stored at between -70°C and -196°C. The composition can be stored at a temperature of -70°C 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 in a frozen state, the composition remains 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 and periodontal bone inflammation. Preferably, the composition comprises IL-4,10,13, VEGF, PDGF, TGFp. Also described is the composition produced by the method described herein.

According to one aspect, there is provided a method of producing a storage stable autologous composition comprising regenerative growth factors and cytokines obtained from platelets, the method comprising the following steps: collecting blood from a mammal or providing blood from a mammal; delivering the blood to a receptacle preferably including an anticoagulant; centrifuging the blood to separate a platelet rich plasma component from the blood; collecting the platelet rich plasma component; passing the platelet rich plasma component through a small pore filter to produce the storage stable autologous composition comprising regenerative growth factors and cytokines derived from platelets; and collecting the storage stable autologous composition, wherein the storage stable autologous composition retains regenerative biological activity for up to at least nine months when stored in a frozen state. According to another aspect, there is provided a storage stable 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-β, IGF1 and VEGF, wherein the storage stable autologous composition retains regenerative biological activity for up to at least nine months when stored in a frozen state.

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 level 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 level of VEGF in the thrombin and mechanically activated PRP.

Figure 6 is a plot of TGF concentration in pg/ml showing a comparison of the level of TGFp in the thrombin and mechanically activated PRP. Figure 7 is a plot of IL-4 concentration in pg/ml showing a comparison of the level of IL-4 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 8 is a plot of IL-10 concentration in pg/ml showing a comparison of the level of IL-10 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 9 is a graph showing a comparison between the level of IL-13 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 10 is a graph showing a comparison between the level of PGDF in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 1 1 is a graph showing a comparison between the level of VEGF in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 12A is a graph showing a comparison between the level of TGF in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

Figure 12B is a graph indicating instability of TGF-βΙ concentration upon different conditions.

Figure 13 is a graph showing a comparison between the level of IGF 1 in the fresh and 9 months stored stable solution of Platelet Rich Plasma Ingredients.

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 nine months when stored preferably at between -70°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 ⁵ , IL-10 ⁶ ' ⁷ , IL-13 ⁸ , PDGF ⁹ , TGF-β ¹⁰ ' ^{1 1} , IGF1 and VEGF. ¹² - ¹³ ' ¹⁴

IL-4,10,13, PDGF, IGF1 , TGF- β 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™.

Preferably, the isolated PRP fraction is harvested and 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 0.15μπι to about 1 μιη. Most preferably the pore size of the filter is about 0.22μπι. An example of a filter that is suitable for carrying out this method is Millipore Corporation, Millex-GP 33mm PES .22um 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 debris is caught in the filter. The liquid composition that is obtained after the PRP fraction passes through the filter is the storage stable autologous composition comprising regenerative growth factors and cytokines derived from platelets.

Preferably, the storage stable autologous composition is then passed a second time through a separate small pore filter having a pore size in the range of about 0.15μηι to about 1 μηι, and preferably about 0.22μιη. 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 next step involves collecting the storage stable autologous composition 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 HEP A 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 -70°C to about - 196°C and most preferably at about -80°C followed by storing for up to 9 months while maintaining acceptable biological activity and therapeutic efficacy of the storage stable autologous composition. The storage stable autologous composition can be frozen and stored while maintaining acceptable biological activity and therapeutic efficacy for at least nine months.

The method of PRP mechanical activation of the present disclosure leads to the same cytokines and growth factors in the compositions as a thrombin activation.

This composition can be stored in a frozen state for at least nine months without decreasing its biological activity and reducing IL-4, IL-10, IL-13, PDGF, TGF-β, IGF1 and VEGF concentrations.

The properties of the storage stable autologous composition 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 lcc 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 0.22μηι. The PRP fraction was passed through a 0.22μιη 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 TGF 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 Throufih Mechanical Activation of Platelets in Fresh Autologous Composition and in Composition Stored for 9 Months at -80°C

The storage stable autologous composition was obtained according to the method of the present disclosure. The composition was divided into aliquots of 2-4cc by carefully drawing of the composition by a sterile syringe and needle. The aliquots were 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 storage stable autologous composition to the level of IL-4 in the storage stable 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 storage stable 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 storage stable autologous composition to the level of IL-10 in the storage stable 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 storage stable 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 storage stable autologous composition to the level of IL-13 in the storage stable 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 storage stable 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 storage stable autologous composition to the level of PGDF in the storage stable 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 storage stable 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 storage stable autologous composition to the level of VEGF in the storage stable 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 storage stable autologous composition is preserved after storage for nine months at -80°C.

Figure 12 A is a plot of TGFP-l concentration in pg/ml showing a comparison of the level of TGFP in the fresh storage stable autologous composition to the level of TGFp-l in the storage stable 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-l in the storage stable autologous composition is preserved after storage for nine months at -80°C.

Figure 12 B is a plot of TGFp-l 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 TGF -l whereas the freezing method (A) preserves normal concentration.

Figure 13 is a plot of IGF1 concentration in pg/ml showing a comparison of the level of IGF1 in the fresh storage stable autologous composition to the level of IGF1 in the storage stable 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 IGF 1 in the storage stable autologous composition is preserved after storage for nine months at -80°C.

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.

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