FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
As amended by the Patents (Amendment) Act, 2005
&
The Patents Rules, 2003
As amended by the Patents (Amendment) Rules, 2006
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION
A growth factor concentrate for treating facial wrinkles
APPLICANTS
Kasiak Research Pvt Ltd, Hoechst House, 17th Floor, Nariman Point, Mumbai 400 021, Maharashtra, India, an Indian company.
INVENTORS
TOTEY, Satish Mahadeorao, 903, Amanda, Hiranadani Eadows. Gladys Alwares Road, Thane (W) 400610, India; SHAH, Kaushal Piyush, B/701, Shree Rajlaxmi Chs, Padma Nagar, Chikuwadi, Borivali (W), Mumbai-400092, India; FONSECA, Lyle Carl, 4b/3 Sangeeta Apartments,Juhu Road, Mumbai-400049, India and MANIYAR, Rachana Rajiv, 405, Chandralok-A, 97, Nepeansea Road, Mumbai-400006, India, all Indian nationals
PREAMBLE TO THE DESCRIPTION
The following specification particularly describes this invention and the manner in which it is to be performed:

FIELD OF THE INVENTION
This invention relates to a growth factor concentrate for treating facial wrinkles.
BACKGROUND OF THE INVENTION
With the advancement of age, wrinkles, grooves, and ptotic tissue become more prominent on a person's face. Superficial wrinkles are largely due to photo damage and resulting solar elastosis. This is characterized by loss of collagen mass in the epidermal-dermal junction and an increased array of elastin whirls in the deeper dermis. Repeated muscle action produces prominent wrinkles and creases in the mimetic areas of facial skin such as the glabella, periorbital skin, nasolabial creases, perioral skin etc. Grooves appear deeper in the nasolabial and marionette zones with the additional feature of fat atrophy. As a result of the loss of fat volume, the static suspensory ligaments become more lax and the face takes on attributes of ptotic jowls, ptotic malar mounds and nasolabial wrinkles. Skeletal changes resulting in decreased height of the maxilla and the mandible occur in the later decades of life and accentuate the above findings. The nasolabial wrinkles, commonly known as "smile lines" or "laugh lines," are facial features. They are the two skin folds that run from each side of the nose to the corners of the mouth. They separate the cheeks from the upper lip. The term derives from Latin nasus for "nose" and labium for "lip".
These wrinkles develop as we age and can be one of the first visible signs of facial aging. That's why nasolabial wrinkles are also one of the first areas of the face targeted by anti-aging or facial rejuvenation procedures. With ageing the fold may become more pronounced and may become cosmetically undesirable to some. The

most significant factors that contribute to the prominence of the fold are excess skin, skin thinning, excess cheek fat, and ptosis of cheek fat.
Methods of addressing facial wrinkles range from excision and re-draping of excess skin to methods of skin resurfacing including dermabrasion, laser ablation, and chemical peels. Each of these modalities has limitations. Implantation of a filler substance into a deficient area is another cosmetic solution. Many materials have been tried for the correction of soft tissue defects and deficits. However, the use of most of these substances has met with difficulties, including impermanence, foreign body reaction, unnatural texture, and possible disease transmission. In addition, most of the products currently available remain relatively expensive.
Small volumes of liquid silicone formerly were used as free injections. Defects intentionally were under-corrected, since the ongoing soft tissue reaction created additional volume. However, the reaction of the host tissue led to late complications including chronic edema, lymphadenopathy, scarring, skin ulceration, skin thinning and discoloration, and siliconomas.
Collagen currently is used widely for treating facial wrinkles. Available collagen consists of purified bovine collagen comprising 95% type 1 collagen and 5% type III collagen. The addition of glutaraldehyde for cross-linking such collagen slows resorption; however, the substance nonetheless eventually is degraded by the host. While collagen works well for the correction of fine lines, results for deeper furrows are less impressive. The major drawback to the use of collagen is the short duration of the response. Within 3 to 6 months the collagen is resorbed completely, and the

improvement is lost. Also, because the collagen is bovine-derived, pre-testing for an allergic response is required.
The Food and Drug Administration (FDA) approved Gore-Tex (solid polytetrafluoroethylene) for facial plastic surgery in 1993. The result is generally predictable because the substance is nonreactive, but the resulting feel of the tissue is somewhat unnatural. The substance is rigid and is not available in an injectable form. In addition, infection remains a potential problem because the substance is a foreign body. Because this substance does not become integrated into the host tissue, it is generally easy to remove.
Newer substances are being marketed. Sheets of human dermis are also available and produce a more natural result. Again, the form of this substance limits its use. Although it is derived from a human source and becomes revascularized by the host, it is not autogenous. Concerns about possible disease transmission have been expressed, although processing appears to control for this. A micronized form of human dermis has been available since 2000. While the semi-liquid form of the substance gives it the versatility of collagen, the substance does not have the longevity of the sheet form. The major impediments to use of this substance remain the high cost and, although initially touted as a permanent solution, the substance is eventually degraded by the host.
Use of injectable poly-L-lactic acid (PLLA) has gained widespread popularity in Europe since its approval for cosmetic correction in 1999. PLLA is a synthetic polymer of lactic acid that is biodegradable, resorbable and biocompatible. The large

particle size of PLLA (40-63 μm) prevents it from being phagocytosed by. macrophages, but it can still be used in needles as fine as 26-gauge. The outcome in patients with a loss of facial volume and contours appears to be very good as judged by physicians and patients, with satisfaction rates reaching as high as 70% and 75%, respectively. One multicenter trial conducted by Laglenne and colleagues involving 110 patients reported minor adverse effects, with only 10% of patients experiencing non-specific ecchymoses and no patients experiencing allergic reactions. However, others have reported many cases of granulomas and inflammatory reactions, which are difficult to treat.
SUMMARY OF THE INVENTION
According to an embodiment of the invention there is provided an intra-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 x lO6 to 5000 x106 human platelets per ml for treating facial wrinkles, 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- (3) 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-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 xlO to 5000 x10 human platelets per ml for treating facial wrinkles, 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-dermal, sub-dermal or topical administration for treatment of facial wrinkles 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 facial wrinkles comprising injecting a growth factor concentrate into the facial wrinkles, wherein the growth factor concentrate is derived from approximately 625 x106 human platelets per ml, the concentrate comprising approximately 500 to 1000 pg/ml of EGF, 10 to 20 pg/ml of VEGF, 10 to 25 pg/ml of basic b-FGF, 20000 to 30000 pg/ml of TGF- β and 60000 to 150000 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. 1A and FIG. IB respectively are photographic representations of a study subjects' nasolabial wrinkles before and at three months after treatment with the GFC as per Example 2.
FIG. 2 shows the Wrinkle Severity Rating Scale (WSRS) scores of study subjects. before treatment and at one month, two months and three months after treatment with the GFC as per Example 2. The wrinkles are scored on a scale of 0-4 with Grade 0 being no nasolabial wrinkles and Grade 4 being deepest nasolabial wrinkles.
FIG. 3 shows the Mean WSRS scores of twenty study subjects, before treatment and at one month, two months and three months after treatment with the GFC as per Example 2. The wrinkles are scored on a scale of 0-4 with Grade 0 being no nasolabial wrinkles and Grade 4 being deepest nasolabial wrinkles.
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 (C6H11Na07); sodium acetate trihydrate, (C2H3Na02.3H20); 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 (C6H11Na07); 368 mg of sodium acetate trihydrate, (C2H3Na02.3H20); 37 mg of potassium chloride, (KG); 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 used 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 625 x106 human platelets per ml.
The growth factor concentrate according to an embodiment of the invention is used for treatment of facial wrinkles including but not limited to nasolabial wrinkles.
According to ah embodiment of the invention, there is further provided a therapeutic composition for intra-dermal, sub-dermal or topical 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. The composition can be in the form of a cream, gel, aqueous solution, spray-aerosol or transdermal patch.
According to an embodiment of the invention, there is still further provided a method of treatment of nasolabial wrinkles comprising injecting a growth factor concentrate into the nasolabial wrinkles, wherein the growth factor concentrate is derived from approximately 625 x 106 human platelets per ml. the concentrate comprising

approximately 500 to 1000 pg/ml of EGF, 10 to 20 pg/ml of VEGF, 10 to 25 pg/ml of basic b-FGF, 20000 to 30000 pg/ml of TGF- β and 60000 to 150000 pg/ml of PDGF-AB. Preferably, 2.5ml of the growth factor concentrate is administered to the facial wrinkles on each side of the face. Preferably, the mode of injection of the growth factor concentrate is the fanning technique.
According to an embodiment of the invention there is also provided a method of preparing an intra-dermally, sub-dermally or topically administrable 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 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 yet another embodiment of the invention there is provided a method of preparing an intra-dermally, sub-dermally or topically administrate 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-dermally. sub-dermally or topically 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-dermally, sub-dermally or topically 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-dermally, sub-dermally or topically 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-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 xlO6 to 5000 x106 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 suspended in an isotonic medium. More preferably, the growth factor concentrate is derived from approximately 625 x lO6 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 x 106 platelets per ml; preferably approximately 625 x 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 9 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-p), 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-lp), Macrophages inhibitory protein-1 alpha (MlP-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 nasolabial wrinkles 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 significant reduction in nasolabial wrinkles.

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 11270 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 dermatological conditions comprising administering topically, intra-dermally or sub-dermally 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 methods above for treatment of human dermato logical conditions. The conditions can include naso-labial wrinkles, facial wrinkles and undesired age-related dermato logical changes.
Preferably, nasolabial wrinkles are treated with the growth factor concentrate derived from approximately 625 x 10 human platelets per ml.
According to another embodiment of the invention, there is provided a therapeutic composition for topical, sub-dermal or intra-dermal 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 cream, gel, aqueous solution, spray-aerosol or transdermal patch.

According to an embodiment of the invention, the GFC is administered by fanning technique of injection wherein a 28 - 30 gauge needle is inserted into the skin, 0.3 ml to 0.4 ml of GFC is injected, the needle is then retracted slightly and the needle reinserted at a different angle and again 0.3 ml to 0.4 ml of GFC is injected. This is done repeatedly and results in a fan-shaped pattern of injections.
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 5 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 15 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 3125 x 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 0.22 micron filter.
Since the final volume of the GFC is 5 ml, and the number of platelets used for preparing the GFC was 3125x105, the effective concentration of platelets used for preparation of the GFC is 625 β 106 platelets per ml.
Example 2
Prospective, multi-centric, open label, randomized, bio-interventional, phase I/IT pilot study to evaluate the safety and efficacy of autologous growth factor concentrate for treatment of nasolabial wrinkles 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 nasolabial wrinkles. The primary objective was to assess the safety of autologous GFC in treatment of subjects with nasolabial wrinkles. The secondary objective was to assess the efficacy of autologous GFC in treatment of subjects with nasolabial wrinkles.

Male and female subjects were recruited in the study. Inclusion and exclusion criteria were as follows:
Inclusion Criteria:
Inclusion of subjects for this study were under the investigator's discretion. However the following criteria were considered.
1. Subjects (male and female), aged 18 to 40 years (both inclusive).
2. Subjects with Grade 3 or Grade 4 nasolabial wrinkles based on the Wrinkle Severity Rating Scale (WSRS) scores of study subjects, wherein the wrinkles are scored on a scale of 0-4 with Grade 0 being no nasolabial wrinkles and Grade 4 being deepest nasolabial wrinkles.
3. Subjects willing to refrain from any other treatment for nasolabial wrinkles.
4. Subjects who were willing to give informed consent and adhere to the study protocol.
Exclusion Criteria:
Exclusion of patients from this study was under the investigator's discretion. However the following criteria were considered
1. Subjects receiving any aesthetic facial therapy 6 months prior to enrolment e.g. Botox, Fillers etc.
2. Subjects with a history of connective tissue disease.
3. Subjects with metabolic or hematopoietic disorders.
4. Subjects with known bleeding disorders such as platelet dysfunction syndrome, thrombocytopenia, hypofibrinogenemia.

5. Subjects with uncontrolled Diabetes mellitus
6. Subjects with prior chemotherapy and radiotherapy.
7. Subjects with immuno-compromised systems
8. Subjects with autoimmune diseases.
9. Female subjects who are pregnant and lactating
10. Subjects who are heavy smokers
11. Subjects with current or past (last 12 months) abuse of alcohol or drugs.
12. Subjects with clinically significant medical or psychiatric disease as determined by the investigator.
13. Subjects unwilling to or unable to comply with the study protocol.
14. Subjects taking concomitant therapy that might interfere with the study results in the investigator's opinion or participating in another trial in the past 30 days.
After an informed consent was taken, the patients were checked for inclusion and exclusion criteria before recruiting for the clinical study and are screened for Infectious Disease markers.
In order to inject the GFC prepared by Example 1 by the farming-technique, one 5 ml dose of GFC prepared as per Example 1 was drawn into a syringe with a 28 to 30G needle. Before injection, the face was cleaned and all the make-up was removed. The nasolabial wrinkle area was cleaned and disinfected thoroughly. A topical anaesthesia cream was applied to the injection area and kept for 20 to 30 minutes to act. The point of injection was marked with a sterile marker pen. The dose was given intra-dermally in the area of the naso-labial wrinkle by the farming-technique. 0.3 ml to 0.4 ml of

GFC was injected, the needle was then retracted slightly and the needle re-inserted at a different angle and again 0.3 ml to 0.4 ml of GFC was injected. This was done repeatedly and resulted in a fan-shaped pattern of injections. A total of 2.5ml was administered to each side of the face in the area of the nasolabial wrinkle by this technique.
All the analysis was done by photographic scores. The study subject's photographs were taken after the recruitment at the designated angle. A visual comparison of the nasolabial wrinkles before administration of the GFC and at three months after administration of the GFC is shown in the photographic representations of FIG. 1A and FIG.IB respectively for a representative study subject. FIG. 2 shows the Wrinkle Severity Rating Scale (WSRS) scores of twenty study subjects, before treatment and at one month, two months and three months after treatment with the GFC as per Example 2. The result of statistical analysis with the mean scores of the twenty study subjects is depicted in FIG, 3, with levels of significance. Analysis was carried out by the Friedman test followed by Dunn's test. The wrinkles are scored on a scale of 0-4 with Grade 0 being no nasolabial wrinkles and Grade 4 being deepest nasolabial wrinkles. Results in FIG. 2 and FIG 3 show that the nasolabial wrinkles reduce significantly after treatment with the GFC prepared by Example 1.
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 can be administered to patients to treat facial wrinkles. 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 floc 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 facial wrinkles. Injection of GFC at the area of facial wrinkles results in a significant reduction in the prominence of facial wrinkles possibly by increasing collagen synthesis and proliferation of fibroblasts. 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 facial wrinkles. 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 facial wrinkles. Growth factors such as Transforming Growth Factor- β (TGF- β) help in proliferation of fibroblasts that in-turn helps in synthesis of collagen and elastin.
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.

WE CLAIM:
1. An intra-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 x 106 to 5000 x106 human platelets per ml for treating facial wrinkles, 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- P) and 200000 to 600000 pg/ml of Platelet Derived growth factor-AB (PDGF-AB).
2. A lyophilized, intra-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 xlO to 5000x10 human platelets per ml for treating facial wrinkles, 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 625 x 106 human platelets per ml.
5. Use of the growth factor concentrate as claimed in claim 1 or 2 for treatment of facial wrinkles.
6. A therapeutic composition for intra-dermal, sub-dermal or topical administration for the treatment of facial wrinkles 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 cream, gel, aqueous solution, spray-aerosol or transdermal patch.
8. A method of treatment of facial wrinkles comprising injecting a growth factor concentrate into the facial wrinkles, wherein the growth factor concentrate is derived from approximately 625 x lO6 human platelets per ml, the concentrate comprising approximately 500 to 1000 pg/ml of EGF, 10 to 20 pg/ml of VEGF, 10 to 25 pg/ml of b-FGF, 20000 to 30000 pg/ml of TGF- p and 60000 to 150000 pg/ml of PDGF-AB.
9. The method as claimed in claim 8, wherein 2.5ml of the growth factor concentrate is administered to the facial wrinkles on each side of the face.
10. The method as claimed in claim 8, wherein the mode of injection of the growth factor concentrate is fanning technique.