WO2014027359A1

WO2014027359A1 - A growth factor concentrate for treating tendinopathies

Info

Publication number: WO2014027359A1
Application number: PCT/IN2013/000263
Authority: WO
Inventors: Satish Mahadeorao Totey; Kaushal Piyush SHAH; Lyle Carl FONSECA; Rachana Rajiv MANIYAR
Original assignee: Kasiak Research Pvt. Ltd.
Priority date: 2012-08-17
Filing date: 2013-04-22
Publication date: 2014-02-20

Abstract

The invention relates to an intra-articularly administrable growth factor concentrate derived from approximately 250 x 10⁶ to 5000 x 10⁶ human platelets per ml for treating tendinopathies. The concentrate comprises approximately 900 to 2000 pg/ml of Epidermal growth factor (EGF), 30 to 300 pg/ml of Vascular Endothelial growth factor(VEGF), 20 to 100 pg/ml of Basic fibroblast growth factor (b-FGF), 40000 to 120000 pg/ml of Transforming growth factor-β (TGF- β) and 200000 to 600000 pg/ml of Platelet Derived growth factor-AB (PDGF-AB).

Description

TITLE OF THE INVENTION

A growth factor concentrate for treating tendinopathies

FIELD OF THE INVENTION

This invention relates to a growth factor concentrate for treating tendinopathies.

BACKGROUND OF THE INVENTION

Tendons connect muscle to bone and allow transmission of forces generated by muscle to bone, resulting in joint movement. Tendon injuries produce considerable morbidity, and the disability that they cause may last for several months despite what is considered appropriate management. Chronic problems caused by overuse of tendons probably account for 30% of all running-related injuries, and the prevalence of elbow tendinopathy in tennis players can be as high as 40%. The basic cell biology of tendons is still not fully understood, and the management of tendon injury poses a considerable challenge for clinicians.

The oxygen consumption of tendons and ligaments is 7.5 times lower than that of skeletal muscles. Their low metabolic rate and well-developed anaerobic energy-generation capacity are essential to carry loads and maintain tension for long periods, reducing the risk of ischemia and subsequent necrosis. However, a low metabolic rate results in slow healing after injury.

Although there is a wide range of tendon pathologies, it appears that the majority of overuse tendinopathies in athletes are due to tendinosis, with collagen degeneration, fibre disorientation, increased mucoid ground substances and an absence of inflammatory cells. Therefore, tendinosis is the end result of a number of etiologic processes with a fairly small spectrum of histological manifestations. The essence of tendinosis is degeneration in tendon cells, collagen fibers, and the subsequent increase in non-collagenous matrix.

Tennis elbow, also known as lateral epicondylitis, follows excessive use of wrist extensors and forearm supinators. Up to 40% of tennis players suffer from it. Tennis elbow affects approximately 1% to 2% of the population and is between 5 to 9 times more common than medial epicondylitis. Although any of the common extensor origin tendons can be involved in this condition, the extensor carpi radialisbrevis tendon is the most commonly involved specific site. Patients most commonly present with lateral elbow pain that frequently radiates into the proximal extensor forearm musculature. Most patients relate symptoms to activities that stress the wrist extensor and supinator muscles, and especially to activities that involve forceful gripping, or lifting of heavy objects.

The present therapies for tendinopathies include eccentric exercise therapy which has been reported to have some effect in prospective, randomized trials in athletic patients. However, a review of 20 published trials found that there was little evidence of a positive effect on clinical outcomes, such as reduction of pain, return to function and patient satisfaction(Woodley BL et al, (2007) Chronic tendinopathy: effectiveness of eccentric exercise. Br J Sports Med 41 : 188- 198). Shock-wave therapy, which is thought to function on the tenocytes to stimulate repair, might be effective in a carefully selected group of patients, although other studies have reported no significant effect (Speed CA (2004) Extracorporeal shock-wave therapy in the management of chronic soft-tissue conditions. J Bone Joint Surg Br 86: 165-171). SUMMARY OF THE INVENTION

According to an embodiment of the invention there is provided an intra-articularly administrable growth factor concentrate derived from approximately 250 * 10⁶ to 5000 x l O⁶ human platelets per ml for treating tendinopathies, the concentrate comprising approximately 900 to 2000 pg/ml of Epidermal growth factor (EGF), 30 to 300 pg/ml of Vascular Endothelial growth factor(VEGF), 20 to 100 pg/ml of Basic fibroblast growth factor (b-FGF), 40000 to 120000 pg/ml of Transforming growth factor-β (TGF- β) and 200000 to 600000 pg/ml of Platelet Derived growth factor-AB (PDGF-AB). According to another embodiment of the invention there is provided a lyophilized, intra- articularly administrable growth factor concentrate derived from approximately 250 x lO⁶ to 5000 x l O⁶ human platelets per ml for treating tendinopathies, the concentrate comprising approximately 900 to 2000 pg/ml of EGF, 30 to 300 pg/ml of VEGF, 20 to 100 pg/ml of b-FGF, 40000 to 120000 pg/ml of TGF- β and 200000 to 600000 pg/ml of PDGF-AB suspended in an isotonic medium along with excipients for lyophilisation.

According to yet another embodiment of the invention there is provided a therapeutic composition for intra-articular administration comprising the growth factor concentrate in combination with supplemental constituents including blood, saline, silver nanoparticles, hyaluronic acid, immuno-modulatory peptides, growth factors, hormones, antibiotics, monoclonal antibodies, recombinant receptors, carriers or combinations thereof.

According to still another embodiment of the invention there is provided a method of treatment of tendinopathies comprising injecting a growth factor concentrate into the lateral epicondyle of the affected region, wherein the growth factor concentrate is derived from approximately 1250 x lO⁶ human platelets per ml, the concentrate comprising approximately 900 to 2000 pg/ml of EGF, 30 to 300 pg/ml of VEGF, 20 to 100 pg/ml of b-FGF, 40000 to 120000 pg/ml of TGF- β and 200000 to 600000 pg/ml of PDGF-AB. It is to be understood that both the foregoing general description and the following detailed description of the present embodiments of the invention are intended to provide an overview or framework for understanding the nature and character of the invention as it is claimed. The accompanying graphical representations are included to substantiate the invention and are incorporated into and constitute a part of this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part ' of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:

FIG. 1 is a graph showing the Patient Rated Tennis Elbow Evaluation (PRTEE) score of study subjects, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 100 with 0 being the best score (no pain) and 100 being the worst score (extreme pain).

FIG. 2 is a graph showing the American Shoulder and Elbow Society (ASES) score of study subjects, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 30 with 0 being the worst score (extreme pain) and 30 being the best score (no pain).

FIG. 3 is a graph showing the tennis elbow pain scale score based on the study subjects assessment of pain on a scale of 0-10, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 10 with 0 being the best score (no pain) and 10 being the worst score (extreme pain).

FIG. 4 is a graph showing the mean PRTEE score of ten study subjects, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 100 with 0 being the best score (no pain) and 100 being the worst score (extreme pain).

FIG. 5 is a graph showing the mean ASES score of ten study subjects, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 30 with 0 being the worst score (extreme pain) and 30 being the best score (no pain).

FIG. 6 is a graph showing the mean tennis elbow pain scale score based on the study subjects assessment of pain on a scale of 0-10, before treatment and at one, two and three months after treatment with the GFC as per Example 2. The total score is out of 10 with 0 being the best score (no pain) and 10 being the worst score (extreme pain).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For simplicity and illustrative purposes, the present invention is described by referring mainly to exemplary embodiments thereof. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one of ordinary skill in the art that the present invention may be practiced without limitation to these specific details. In other instances, well known methods have not been described in detail so as not to unnecessarily obscure the present invention.

In the context of the invention, the term "growth factor concentrate" or "GFC" as used in the specification refers to a standardized concentration of growth factors prepared according to an embodiment of. the invention, the growth factors being derived from the cryo-stimulation of platelets which have been counted, the platelets being sourced from human blood. The GFC also contains cytokines, chemokines, adhesive proteins and other modulatory peptides.

Also, "multiple electrolyte isotonic solution" as used in the specification in the context of the invention comprises sodium chloride, (NaCl); sodium gluconate (C₆HnNa0₇); sodium acetate trihydrate, ( ^HsNaC 'SFbO); potassium chloride, (KC1); and magnesium chloride, (MgCl2^»6H₂0) and human serum albumin. Preferably, each 100 ml of multiple electrolyte isotonic solution contains 526 mg of sodium chloride, (NaCl); 502 mg of sodium gluconate (C₆HnNa0₇); 368 mg of sodium acetate trihydrate, (C₂H₃Na0₂'3H₂0); 37 mg of potassium chloride, (KC1); and 30 mg of magnesium chloride, (MgCl2'6H₂0) and 1 to 5% of human serum albumin. The pH is adjusted with sodium hydroxide to 7.4 (or 6.5 to 8.0), sterile filtered and tested for endotoxin.

Further, the term "platelet free plasma" as used in the specification in the context of the invention comprises the supernatant collected from plasma that has been centrifuged at about 17610 g and then sterile filtered. The term "platelet poor plasma" or "PPP" as used in the specification in the context of the invention comprises supernatant collected from whole blood that has been centrifuged at about 2720 g. Preferably, the isotonic medium in the growth factor concentrate is multiple electrolyte isotonic solution, plasma, platelet free plasma, platelet poor plasma or a combination thereof.

Preferably, the concentrate is derived from approximately 1250 x l O⁶ human platelets per ml.

The growth factor concentrate according to an embodiment of the invention is used for treatment of tendinopathies, particularly in the treatment of tennis elbow. The method of treatment provided by the present invention preferably involves injecting upto 5ml of the growth factor concentrate into the lateral epicondyle region of the tennis elbow.

The therapeutic composition according to an embodiment of the invention can be in the form of a gel or aqueous solution.

According to an embodiment of the invention there is further provided a method of preparing an intra-articularly administrate growth factor concentrate derived from human platelets comprising the following steps:

a. suspending human platelets in multiple electrolyte isotonic solution; b. snap-freezing the suspension;

c. thawing the frozen suspension; and

d. sterile-filtering the suspension

wherein, a fixed number of platelets is suspended in a fixed volume of multiple electrolyte isotonic solution to obtain the required concentration of growth factors growth factor concentrate,

snap-freezing of the suspension is carried out at a temperature of -120°C to -200°C, thawing of the frozen suspension is carried out at 25°C to 37°C, and cellular debris are separated from the thawed suspension and the resultant suspension of growth factors is diluted with an isotonic medium before sterile-filtering.

According to yet another embodiment of the invention there is provided a method of preparing an intra-articularly administrable growth factor concentrate derived from human platelets comprising the following steps:

a. suspending human platelets in upto 5ml of plasma;

b. snap-freezing the suspension;

c. thawing the frozen suspension; and

d. sterile-filtering the suspension

wherein, a fixed number of platelets is suspended in a fixed volume of plasma to obtain the required concentration of growth factors in the growth factor concentrate, snap-freezing of the suspension is carried out at a temperature of -120°C to -200°C, thawing of the frozen suspension is carried out at 25°C to 37°C, and

cellular debris are separated from the thawed suspension and the resultant suspension of growth factors is diluted with an isotonic medium before sterile-filtering.

According to a further embodiment of the invention there is provided a method of preparing an intra-articularly administrable growth factor concentrate derived from human platelets comprising the following steps:

a. suspending human platelets in an isotonic medium;

b. snap-freezing the suspension;

c. thawing the frozen suspension; and

d. sterile-filtering the suspension wherein, a fixed number of platelets is suspended in a fixed volume of the isotonic medium to obtain the required concentration of growth factors in the growth factor concentrate;

the snap-freezing is carried out at a temperature of - 120°C to -200°C;

the thawing is carried out at 25°C to 37°C; and

cellular debris are separated from the thawed suspension and the resultant suspension of growth factors is diluted with the isotonic medium before sterile-filtering and lyophilized with excipients after sterile-filtering,

provided that where the isotonic medium in step (a) is plasma, the volume of plasma does not exceed 5 ml.

According to yet another embodiment of the invention there is provided an intra-articularly administrable growth factor concentrate derived from a fixed number of human platelets, the concentrate comprising the required concentration of growth factors suspended in an isotonic medium.

According to still another embodiment of the invention there is provided a lyophilized, intra- articularly administrable growth factor concentrate derived from human platelets, the concentrate comprising the required concentration of growth factors suspended in an isotonic medium along with excipients for lyophilisation.

According to yet another embodiment of the invention there is provided a dosage of an intra- articularly administrable growth factor concentrate derived from approximately 250 * 10⁶ to 5000 x l O⁶ human platelets per ml, the concentrate comprising approximately 900 to 2000 pg/ml of Epidermal growth factor (EGF), 30 to 300 pg/ml of Vascular Endothelial growth factor(VEGF), 20 to 100 pg/ml of Basic fibroblast growth factor (b-FGF), 40000 to 120000 pg/ml of Transforming growth factor-β (TGF- β) and 200000 to 600000 pg/ml of Platelet Derived growth factor-AB (PDGF-AB) suspended in an isotonic medium. More preferably, the growth factor concentrate is derived from approximately 1250 ^χ ΐθ⁶ human platelets per ml

Freshly collected platelets or fresh blood can be collected from donors or even blood banks for large scale manufacturing of GFC. The blood is preferably transported to the central processing laboratory at 20-24°C in a transportation box. Blood can alternatively be collected from donors requiring treatment with the GFC. Blood may also be collected from other mammalian species such as horse, dog, cat, buffalo, cow, sheep, goat, rodents etc. from either jugular vein or the cephalic vein or femoral vein. One part of the blood sample collected is preferably routinely processed for complete blood count (CBC), and rapid infectious disease marker testing for Human Immunodeficiency Virus (HIV- 1,2), Hepatitis B virus (HBV), Hepatitis C Virus (HCV), Venereal disease research laboratory (VDRL) tests. The remaining part of the blood collected is preferably sent to a class B environment clean-room for further processing to yield the GFC. The clean room temperature is preferably maintained at 22°C with a relative humidity of 55 %.

Stability of the platelets in the whole blood (in terms of growth factor levels) was checked for different temperatures and time points and it was found that platelets in whole blood are stable between 15 to 30°C until 24 hours for the purpose of GFC preparation i.e. the growth factor levels measured using ELISA after recovering platelets at different temperatures and time points remained stable upto 24 hours.

According to an embodiment of the invention, the blood sample to be used for obtaining the GFC is centrifuged at 109 g to 680 g, preferably at about 382 g for 15 minutes at 22°C for isolation of platelets. After centrifugation, three layers are observed: a top layer of yellow coloured platelet rich plasma (PRP), a middle layer of white blood corpuscles (WBC) and the bottom layer of red blood corpuscles (RBC). The top layer is aspirated carefully to maximize platelet yield, while ensuring that no WBCs are picked up, and placed in another sterile centrifuge tube. The platelet rich plasma collected is then centrifuged at 680 to 3442 g, preferably at about 2720 g for 10 minutes. This separates the PRP into a platelet pellet and a platelet poor plasma (PPP) supernatant. The entire PPP is collected in a sterile centrifuge tube and stored at room temperature for later use. Platelets of any desired concentrations could be prepared in this manner which is not possible in other known devices for obtaining PRP. For autologous use, the platelet pellet is re-suspended in the appropriate amount of isotonic medium. Preferably, the isotonic medium is platelet free plasma, platelet poor plasma, multiple electrolyte isotonic solution or combinations thereof. For allogeneic purposes, the concentrated platelet pellet can be re-suspended in 1 to 10 ml of multiple electrolyte isotonic solution or platelet free plasma which is tested for ABO compatibility before use. The platelets are then counted and further isotonic medium can be added so that the platelet numbers are adjusted to the count of approximately 250 to 5000 ^χ 10⁶ platelets per ml; preferably approximately 1250 ^χ 10⁶ platelets per ml. This platelet suspension is then subjected to a physiological activation by freezing at - 196°C. The centrifuge tube containing the concentrated platelet suspension is placed in liquid nitrogen for 120 seconds and then subjected to a rapid thawing. Rapid thawing is done at 37°C for 120 seconds. Just one freeze-thaw cycle is sufficient to physiologically activate the platelets and causes lysis of the platelet membranes. This freeze-thaw cryo-stimulates the platelets to release growth factors. The suspension can then be mixed with 4 to 14 ml of PPP removed at the earlier step or platelet free plasma or multiple electrolyte isotonic solution and subjected to high speed centrifugation at a speed of 17610 g for 30 minutes. The last centrifugation is critical to remove all the plasma membranes or membrane antigens of platelets or debris so as to obtain an acellular solution. After high speed centrifugation supernatant is aspirated and transferred to another sterile tube. The GFC is amenable to lyophilisation after mixing the GFC with 2 to 10% mannitol, sucrose and/or glycine added to the GFC as bulking agent/lyoprotectant for lyophilisation. The lyophilised product is sealed with a flip off cap for clinical application and is stable for more than one year at 4°C. Lyophilised GFC can be reconstituted with 5-15 ml of multiple electrolyte isotonic solution. Sterile water for injection can also be used as a diluent for reconstitution, with 1% human serum albumin. Blood type matched plasma can also be used as a diluent. In an embodiment of the invention, the platelet pellet is suspended in 1ml of multiple electrolyte isotonic solution and after the freeze-thaw, the thawed solution is then mixed with 4 ml of plasma which had been removed from the upper layer at the end of the second centrifugation.

In a preferred embodiment of the invention, the multiple electrolyte isotonic solution is supplemented with pharmaceutically acceptable excipients. Preferably, the pharmaceutically acceptable additives are selected from a group comprising of Acid Citrate Dextrose-solution A (ACD-A), Ethylenediaminetetraacetic acid (EDTA), and Citrate phosphate dextrose adenine (CPDA). GFC comprises a combination of Growth Factors such as Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-beta (TGF-β), basic fibroblast growth factor (bFGF), Insulin like growth factor- 1 (IGF-1), Hepatocyte Growth Factor (HGF), Platelet derived growth Factor- (PDGF-AA), (PDGF-AB), (PDGF-BB); Cytokines like RANTES, Interleukin-1 beta (IL-Ι β), Macrophages inhibitory protein- 1 alpha (ΜΓΡ- la), GRO- alpha, ENA-78, MCP-3, NCP, IGFBP-3; Basic Proteins like Platelet factor 4 (PF-4), Endostatin, PBP, Connective tissue activating peptide(CTAP), Neutrophil activating peptide (NAP); adhesive proteins such as ECGF, Plasminogen activator inhibitior-1, Laminin-8, Fibrinogen, Fibronectin, Thrombospondin and antimicrobial agents like Thrombocidin. These growth factors and other proteins have regenerative properties and help in initiating the healing process.

The GFC product was tested for endotoxin so as to confirm that the product was safe and free from bacterial contamination. The GFC product was also tested for infectious diseases like HIV, HCV, HBV, Syphilis etc and was found to be disease-free and safe for clinical applications. Clinical studies on tendinopathies were undertaken for the GFC prepared by the method of the present invention. In the clinical trials being conducted, administration of GFC led to a speeding up recovery from tendinopathies.

It is within the scope of the invention to use other human cell types i.e. other than platelets as well, to yield the GFC according to the method of the invention. It is also within the scope of the invention to use other mammalian cells or placenta to yield the GFC according to the method of the invention.

Preferably, the snap-freezing is done in liquid nitrogen or in liquid helium. The thawing can be done in a sterile water bath at 37°C. The isotonic medium can be multiple electrolyte isotonic solution, plasma, platelet free plasma, platelet poor plasma or a combination thereof. The multiple electrolyte isotonic solution can be supplemented with pharmaceutically acceptable excipients. Preferably, the excipients include mannitol, sucrose, glycine or combinations thereof. Preferably, the cellular debris are removed from the thawed suspension by centrifuging the thawed suspension at 1 1270 g to 17610 g for 25 to 35 minutes and isolating the supernatant.

The platelets of step (a) of the method can be obtained by plateletpheresis, or by centrifugation of whole blood. The platelets of step (a) are preferably obtained by:

a. centrifuging at least 10 ml of anticoagulated human blood at 109 g to 680 g for 5 to 20 minutes;

b. isolating the top-most layer containing platelets and centrifuging the same at 680 g to 3442 g for 5 to 15 minutes; and

c. isolating the plasma-free pellet of platelets obtained at the end of step (b).

Still preferably, the centrifugation in step (a) is carried out at 382 g for 15 minutes and the centrifugation in step (b) is carried out at 2720 g for 10 minutes.

According to an embodiment of the invention, there is provided a method of treating orthopedic, conditions comprising administering intra-articularly the growth factor concentrate prepared by any of the methods above.

According to another embodiment of the invention, there is provided use of the growth factor concentrate prepared by any of the method s above for treatment of human orthopedic conditions. The conditions can include tennis elbow and other joint related disorders like Achilles tendon.

Preferably, tennis elbow is treated with the growth factor concentrate derived from approximately 1250 x l O⁶ human platelets per ml. According to another embodiment of the invention, there is provided a therapeutic composition for intra-articular administration comprising the growth factor concentrate prepared by any of the methods above in combination with supplemental constituents including blood, saline, silver nanoparticles, hyaluronic acid, immuno-modulatory peptides, growth . factors, hormones, antibiotics, monoclonal antibodies, recombinant receptors, carriers or combinations thereof. The composition can be in the form of a gel or aqueous solution.

According to another embodiment of the invention, there is provided a method of treatment of tennis elbow wherein GFC derived from approximately 1250* 10⁶ human platelets per ml is administered into the lateral epicondyle region of the elbow. The administration is preferably by injection which is preferably guided by ultrasonography.

In order that those skilled in the art will be better able to practice the present disclosure, the following examples are given by way of illustration and not by way of limitation.

Example 1

Human blood was withdrawn into vacutainers after getting informed consent from the patient. About 25-35 ml blood is collected in two types of vacutainers: 25 ml in vacutainers containing ACD-A for preparing GFC and 5-10 ml blood in EDTA tubes for infectious disease marker testing and complete blood parameter testing. Minimum 25 ml of blood was taken for 10 ml of GFC preparation. Blood was transported at 15 to 30°C preferably at 22°C within 4 hours of withdrawal. The first centrifugation was done at 382 g for 15 minutes since platelet recovery at 382 g for 1 minutes is optimum with a loss of only 8-10% platelets. Platelet loss is significantly greater at lower or higher centrifugation speeds. Upon completion of first centrifugation three layers were formed. At the bottom were packed red blood cells, in the middle were leukocytes and the upper layer had plasma containing platelets. Plasma containing platelets was aspirated and transferred in another sterile tube. The second centrifugation was done at 2720 g for 10 minutes since centrifugation at 2720 g for 10 minutes results in platelet recovery of almost 99.5%. The upper layer had plasma and at the bottom was a packed platelet pellet. The entire plasma (PPP) was removed and placed in a sterile tube and stored at room temperature for later use. Platelet pellet was suspended in 1 ml of PPP. Platelets were then counted and the concentration of platelets per ml of plasma was noted to be 6250>< 10⁶. The solution was then snap-frozen in liquid nitrogen at -196°C for 2 minutes. The frozen solution was then rapidly thawed in a water bath at 37°C for 2 minutes. In order to standardize the concentration of growth factors in the solution, the thawed solution was then mixed with 4 ml of PPP. This solution was then transferred to another sterile tube and subjected to high speed centrifugation at 17610 g for 30 minutes. The supernatant containing the growth factors was collected and sterile filtered through a 0.22micron filter.

Since the final volume of the GFC is 5 ml, and the number of platelets used for preparing the GFC was 6250x l0⁶, the effective concentration of platelets used for preparation of the GFC is 1250 l0⁶ platelets per ml. Example 2

Prospective, multi-centric, open label, randomized, bio-interventional, phase lill pilot study to evaluate the safety and efficacy of autologous Growth Factor Concentrate for treatment of tennis elbow was carried out. The objective of this pilot study was to demonstrate the potential of autologous GFC prepared by Example 1 for the treatment of subjects with tennis elbow. The primary objective was to assess the safety of autologous GFC in treatment of subjects with tennis elbow. The secondary objective was to assess the efficacy of autologous GFC in treatment of subjects with tennis elbow.

Male and female subjects were recruited in the study. Inclusion and exclusion criteria were as follows:

Inclusion Criteria:

1. Subjects with clinical diagnosis of tennis elbow within the last 3 months

2. Subjects both male and female, aged 18-60 years (both inclusive)

3. Subjects with doppler activity on Ultrasonography

4. Subjects revealing diffuse pain with provocative manoeuvres

5. Subjects with lateral elbow pain that is maximal over the lateral epicondyle and increase with pressure on the lateral epicondyle and resisted dorsiflexion and/or middle finger

6. Subjects willing to refrain from any other concomitant conservative treatment modality including NSAID (must be stopped for at least one week prior to enrolment ih the study)

7. Subjects who are willing to give informed consent and adhere to the study protocol

Exclusion Criteria:

1. Subjects aged <18 and >60 years

2. Subjects with clinical suspicion of insertion disorders

3. Subjects with presence of wounds around the elbow

4. Subjects with pain in hand or shoulder/neck in the same arm being treated.

5. Subjects with suspicion of hyperlipidemia

6. Subject with complex regional pain syndrome 7. Subjects with history of Cervical neuropathy or other nerve pathology

8. Subjects with history of RA, PVD, sarcoidosis, metabolic disease such as gout, frozen shoulder, inflammatory arthropathy, chronic dislocation, spondylarthropathy avascular necrosis, metabolic bone disorders or evidence of intra articular arthritis

9. Subjects with history of connective tissue disease

10. Subjects with history of malignancies

1 1. Subjects with autoimmune diseases

12. Subjects with immuno-compromised system

13. Subjects on Anti-coagulant therapy or blood thinning medicines like Aspirin 14. Subjects taking concomitant therapy that might interfere with the study results in investigators opinion or who had concomitant other injury of the tennis elbow tendons.

15. Subjects who have received treatment with corticosteroid injections within the last 6 months

16. . Subjects who have received prior platelet rich plasma injection

17. Subjects with clinically significant medical or psychiatric disease as determined by the investigator.

18. Female subjects who are pregnant or lactating.

19. Subjects who are heavy smokers

20. Subjects with current or past (last 12 months) abuse of alcohol or drugs

21. Subjects unwilling to or unable to comply with the study protocol

22. Subjects participating in another trial in the past 30 days

23. Condition that prevents the patients from executing an active exercise program.

After informed consent was taken, the patients were checked for inclusion and exclusion criteria before recruiting for the clinical study and were screened for Infectious Disease markers. The GFC prepared by Example 1 was administered with the aid of ultrasonography (USG). USG guided 23 to 24 G needle for administration of GFC was injected into the lateral epicondyle region of the elbow. A volume of 3 ml to 5 ml of GFC was injected by this method into the in the lateral epicondyle region.

All the analysis was done by three different scoring techniques. Results shown in FIG. 1 , FIG. 2 and FIG. 3 indicate that pain due to tennis elbow reduced significantly at two months and three months after treatment with the GFC prepared by Example 1. Statistical analysis was carried out by the Friedman Test followed by Dunn's Test, as shown in FIG. 4, FIG. 5 and FIG. 6. It is evident that pain due to tennis elbow continued to reduce even at three months after treatment and this reduction was statistically significant.

As is evident from the above results, the GFC of the present invention is derived from predetermined numbers of platelets yielding proportionate amounts of growth factors which in turn serves to provide consistent clinical results. Fixed dosages of GFC are administered to patients to treat tendinopathies. Further, less than 10% platelets are lost by the method of the invention. Also, the platelets are physiologically activated without incorporating any additional materials or chemical substances, like calcium chloride or bovine thrombin, and hence it is safe. Also, the single freeze-thaw makes the process less time consuming and more suitable for large scale production and the growth factors are not denatured as in the case of multiple freeze-thaws. Moreover, some clinical indications need a large amount of growth factors but some indications need very little growth factors since the presence of growth factor receptors vary from one cell type to another and one indication to another. Therefore, the present invention provides GFCs that can have standardized concentrations which can be diluted as per the requirement to make it suitable for specific clinical indications. Another benefit of the present method is that multiple doses of GFC can be prepared from a single blood draw and hence this method is cost effective and not time consuming. Further, GFC does not show any flocculation on long-term storage for upto six months at -20°C. A small floe is sometimes seen which generally gets dissolved within 2-3 minutes at room temperature and hence it is possible to use GFC as an "off the shelf product which can be stored for upto six months without any problem of losing its potency. Also, since the GFC is acellular and devoid of plasma membranes or other antigenic materials, it does not elicit any immune reactions or formation of allo-antibodies. GFC can optionally be made plasma free so that it can be used as a therapeutic agent without any problem of ABO incompatible plasma that may cause immune reactions. GFC is also amenable to lyophilisation so that the GFC can be stored at room temperature or in a 4°C refrigerator without any degradation for more than one year. Further, lyophilized GFC can be made into a cream, gel, aqueous solution, spray-aerosol or transdermal patch. All in all, GFC is a natural i.e. non- recombinant product and the method provided by the present invention for production of GFC is economical. Further, GFC shows improved clinical outcomes due to the significantly higher level of growth factors in GFC as compared to multiple freeze-thawed HPL prepared by known methods. GFC also serves as a personalised therapy for patients requiring specific concentrations of GFC to be administered as the GFC can be prepared in any desired concentration. GFC contains therapeutic level of growth factors that is specifically suitable for treating tendinopathies. Injection of 3 to 5 ml of GFC derived from 1250* 10⁶ human platelets per ml into the lateral epicondyle region of the elbow results in a significant reduction in the pain possibly by modulating the local immune response and inflammatory activity within the joint microenvironment and jumpstarting tissue repair. GFC derived from the platelets contains several important growth factors, cytokines, chemokine, anti-microbial agents and basic proteins and has very good therapeutic potential for various indications including tendinopathies. As is evident, the dosage of GFC administered and method of administration according to an embodiment of the present invention result in significant reduction of the pain caused by tennis elbow. Growth factors such as Transforming Growth Factor-β (TGF-β) help in cell proliferation that in-turn helps in tissue repair. Vascular Endothelial Growth Factor (VEGF) helps in strengthening vasculature and the supply of blood to the site of injury and hence the GFC proves useful for the treatment of tendinopathies by modulating inflammation.

What has been described and illustrated herein are preferred embodiments of the invention along with some of their variations. The terms, descriptions and figures used herein are set forth by way of illustration only and are not meant as limitations. Those skilled in the art will recognize that many variations are possible within the spirit and scope of the invention, which is intended to be defined by the claims in the complete specification— and their equivalents— -in which all terms are meant in their broadest reasonable sense unless otherwise indicated.

Claims

CLAIMS:

1. An intra-articularly administrable growth factor concentrate derived from approximately 250 x l O⁶ to 5000 * 10⁶ human platelets per ml for treating tendinopathies, the concentrate comprising approximately 900 to 2000 pg/ml of Epidermal growth factor (EGF), 30 to 300 pg/ml of Vascular Endothelial growth factor(VEGF), 20 to 100 pg/ml of Basic fibroblast growth factor (b-FGF), 40000 to 120000 pg/ml of Transforming growth factor-β (TGF- β) and 200000 to 600000 pg/ml of Platelet Derived growth factor- AB (PDGF-AB).

2. A lyophilized, intra-articularly administrable growth factor concentrate derived from approximately 250 x l O⁶ to 5000 x l O⁶ human platelets per ml for treating tendinopathies, the concentrate comprising approximately 900 to 2000 pg/ml of EGF, 30 to 300 pg/ml of VEGF, 20 to 100 pg/ml of b-FGF, 40000 to 120000 pg/ml of TGF- β and 200000 to 600000 pg/ml of PDGF-AB suspended in an isotonic medium along with excipients for lyophilisation.

3. The concentrate as claimed in claim 2 wherein, the isotonic medium is multiple electrolyte isotonic solution, plasma, platelet free plasma, platelet poor plasma or a combination thereof.

4. The concentrate as claimed in claim 1 or 2 derived from approximately 1250 x l O⁶ human platelets per ml.

5. Use of the growth factor concentrate as claimed in claim 1 or 2 for treatment of tennis elbow.

6. A therapeutic composition for intra-articular administration comprising the growth factor concentrate as claimed in claim 1 or 2 in combination with supplemental constituents including blood, saline, silver nanoparticles, hyaluronic acid, immuno-modulatory peptides, growth factors, hormones, antibiotics, monoclonal antibodies, recombinant receptors, carriers or combinations thereof.

7. The composition as claimed in claim 6 wherein, the composition is in the form of a gel or aqueous solution.

8. A method of treatment of tennis elbow comprising injecting a growth factor concentrate into the lateral epicondyle region of the tennis elbow, wherein the growth factor concentrate is derived from approximately 1250 x lO⁶ human platelets per ml, the concentrate comprising approximately 900 to 2000 pg/ml of EGF, 30 to 300 pg/ml of VEGF, 20 to 100 pg/ml of b-FGF, 40000 to 120000 pg/ml of TGF- β and 200000 to 600000 pg/ml of PDGF-AB.

9. The method of treatment as claimed in claim 8, wherein upto 5ml of the growth factor concentrate is injected into the lateral epicondyle region of the tennis elbow.