WO2014027361A1

WO2014027361A1 - A growth factor concentrate for treating periorbital hyperpigmentation

Info

Publication number: WO2014027361A1
Application number: PCT/IN2013/000265
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-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 x 10⁶ to 5000 x 10⁶ human platelets per ml for treating periorbital hyperpigmentation. 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 periorbital hyperpigmentation

FIELD OF THE INVENTION

This invention relates to a growth factor concentrate for treating periorbital hyperpigmentation.

BACKGROUND OF THE INVENTION

Periorbital hyperpigmentation (POH) is also known as under eye dark circles or dark circles. It is an aesthetic facial concern characterized by alterations in the colour of the upper and lower eyelids. POH is characterized by hyper-chromic macules and patches around the eyes. Possible causative factors that have been proposed include excessive constitutional pigmentation, thin and translucent lower eyelid skin, shadowing due to skin laxity, and venous congestion. The skin over the lower eyelid is thinner and looser than other sites because it contains less collagen, elastin, and glycosaminoglycans. It has been reported that darkening around the eyes is caused by a process of dermal melanization and / or post-inflammatory hemodynamic congestion. Melanin and haemoglobin are the dominant chromophores of human skin and their local accumulation promotes changes in skin colour. Evidence suggests that an important mechanism involved in the development of POH is related to sluggish blood flow. It is known that the skin of the eyelids is the thinnest in the body, and conditions such as congestion, hyperaemia, or any other circulatory alterations are readily reflected at the surface. Both congenital and environmental factors, including ultraviolet radiation, chronological aging, physical and emotional stress, allergic and atopic reactions, exogenous or even unbalanced endogenous oestrogen are involved in the cutaneous disturbance, and all these variables lead to the release of inflammatory mediators affecting vascular permeability. POH is a significant cosmetic concern for both female and male patients. Although it does not cause morbidity, it can influence the quality of life from the medical point of view. Dark circles around the eyes are reported to cause a negative impact on social life and on one's emotional state, causing loss of confidence and lowered self-esteem.

Current available treatments for POH are topical creams, topical bleaching, lotions, vitamins, neutraceuticals, chemical peels, periorbital lipotransfer, autologous fat transplantation, and lasers; however, none of them have a satisfactory outcome. US 2012/01 15792 Al discloses treatment of POH by administration of a botulinum toxin type-A to a patient. The botulinum toxin can be administered to the vicinity of an eye of a patient with POH. It is also shown that botulinum neurotoxins are able to change local vascular perfusion by their action on neuro-secretary substances that helps in enhancing blood flow, lymphatic drainage and in turn decreases the pooling blood and extravascular fluid in the periorbital region.

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 l O⁶ to 5000 x l O⁶ human platelets per ml for treating periorbital hyperpigmentation, 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- dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 ^χ ΐθ⁶ to 5000 * 10⁶ human platelets per ml for treating periorbital hyperpigmentation, 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 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 periorbital hyperpigmentation comprising :

a. applying a growth factor concentrate on the hyperpigmented region of the skin under the eye; and

b. rolling a skin micro-needle on the said region to effect infusion of the said concentrate into the skin of the said region,

wherein the growth factor concentrate is derived from approximately 1250 ^χ 10⁶ 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.

Alternatively, the said growth factor concentrate can be injected into the orbicularis oculi muscle. 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 subject's POH before treatment and at 3 months after treatment with a single dose of the GFC as per Example 2.

FIG. 2 is a graph showing the Von Luschan's Chromatic Scale (VLCS) scores for POH of twelve study subjects, before treatment and at one month, two months and three months after treatment with a single dose of the GFC as per Example 2. The shade of the POH is scored on a scale of 1-36 as per the VLCS with 1 being the lightest skin colour and 36 being the darkest skin colour. FIG. 3 is a graph showing the mean VCLS scores of twelve study subjects, before treatment and at one month, two months and three months after treatment with a single dose of the GFC as per Example 2. The shade of the POH is scored on a scale of 1 -36 as per the VLCS with 1 being the lightest skin colour and 36 being the darkest skin colour.

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 (CeHnNaO_?); 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 (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.

Also, the term "baseline" as used in the specification in the context of the invention refers to the VLCS score of a study subject prior to administration of GFC according to an embodiment of the invention. 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 ^χ 10⁶ human platelets per ml. The growth factor concentrate according to an embodiment of the invention is used for treatment of periorbital hyperpigmentation.

The therapeutic composition according to an embodiment of the invention can be in the form of a cream, gel, aqueous solution, spray-aerosol or transdermal patch. Preferably, a single dose of the growth factor concentrate is administered for the treatment of POH, the dose comprising 5ml of the growth factor concentrate, wherein 2.5ml is administered to the hyperpigmented region of the skin under each eye. Administration can be done by skin microneedling or by injection into each orbicularis oculi muscle.

According to another 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 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 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-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 1000 ^χ 10⁶ to 1500 ^χ ΐθ⁶ 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 1250 x 10⁶ 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-rpom 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 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 a§ ko 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), Ethylehediaminetetraacetic 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-1 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 periorbital hyperpigmentation 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 decrease in periorbital hyperpigmentation. 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 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 dermatological conditions. The conditions can include periorbital hyperpigmentation.

Preferably, periorbital hyperpigmentationis treated with the growth factor concentrate derived from approximately 1250 x lO⁶ 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 injection or by skin micro-needling. Skin micro-needling is an alternative rriode of administering the GFC for treating POH. Skin micro-needling is also known as derma rolling, dermaroller, skin rolling or collagen induction therapy (CIT). Micro-needling is a process carried out with a roller that is covered with numerous tiny needles. These needles penetrate the upper layer of the skin to a depth of up to 2mm (depending on the needle length used). This process has the benefit of delivering GFC and massively improves the penetration of GFC effectively through the top layer (epidermis) of the skin. As^' the GFC is absorbed better, its performance is boosted and this leads to better results, leading to healthier, smoother, softer and more radiant skin.

Micro-needling creates a very minor (almost microscopic) trauma which lasts for a short period of time and does not result in bruising or swelling or bleeding in the treated area in the way that chemical peels, laser therapy or microdermabrasion normally do. A micro-needle roller can be used to infuse the GFC into the dermis in order to intensify the natural wound cascade because of the high concentration of growth factors and also help in penetration of the GFC at the dermal layer of the periorbital skin through the creation of multiple micro channels. Micro-needling was found to be an effective, versatile, cost effective delivery system for the GFC prepared by Example 1 as it induced tissue repair by infusing the GFC.

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 1_.5 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 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 5ml, and the number of platelets used for preparing the GFC was 6250>< 10⁶, the effective concentration of platelets used for preparation of the GFC is 1250x l0⁶ platelets per ml. Example 2

Prospective, multi-centric, open label, randomized, bio-interventional, phase I/II pilot study to evaluate the safety and efficacy of autologous growth factor concentrate for treatment of POH 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 POH. The primary objective was to assess the safety of autologous GFC in treatment of subjects with POH. The secondary objective was to assess the efficacy of autologous GFC in treatment of subjects with POH.

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

Inclusion Criteria:

Inclusion of patients for this study will be under the investigator's discretion. However the following criteria will be considered.

1. Subjects (male and female), aged 18 to 55 years (both inclusive) with POH.

2. Subject willing to refrain from any other treatment of POH during the entire study

duration.

3. Subjects who are willing to give informed consent and adhere to the study protocol. Exclusion Criteria:

Exclusion of patients from this study will be under the investigator's discretion. However the following criteria will be considered

1. Subjects aged <18 and > 55 years 2. Subjects undergoing any aesthetic facial therapy 6 weeks prior to enrolment.

3. Subjects with a history of connective tissue disease.

4. Subjects with metabolic or hematopoietic disorders.

5. Subjects with known bleeding disorders such as platelet dysfunction syndrome, thrombocytopenia, hypofibrinogenemia.

6. Subjects who have received prior chemotherapy and radiotherapy

7. Subject with known diabetes mellitus

8. Subject with immune-compromised systems.

9. Subjects with autoimmune diseases.

10. Female subjects who are pregnant or lactating.

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

12. Subjects who are heavy smokers.

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

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

15. Subjects taking concomitant therapy that might interfere with the study results ih the investigator's opinion or participating in another trial in the past 30days.

After 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.

The skin micro-needling process was used to administer the GFC. Prior to treatment all make-up and cosmetic creams were removed. The patient's skin was then cleaned with a topical antiseptic followed by sterile saline to remove all oils on the skin's surface, so as to allow the anaesthetic cream to penetrate effectively. A topical anaesthetic cream was applied to the injection area and kept for 20 to 30 minutes to act. The GFC was applied on the skin. A micro-needle roller (0.25mm to 0.5mm needle length) was used to infuse the GFC prepared in Example 1. The micro-needling roller was rolled forward and backward and in the crisscross direction repeatedly till a total of 5 ml of GFC was used up for the treatment (2.5 ml under each eye).

All the analysis was done by photographic scores. The study subject's photographs were taken after the recruitment at the designated angle. Photographs were taken of Front Face, Left side face, and Right side face. The skin colour was measured by VLCS and scored. The skin colour was measured before injection and at one month, two months and three months after injection. FIG.1A and FIG. IB respectively are photographic representations of one study subject's POH before treatment and at three months after treatment with a single dose of the GFC as per Example 2. The scores of twelve study subjects are depicted graphically in FIG.2 which shows that periorbital hyperpigmentation is significantly reduced from the baseline. The result of statistical analysis with the mean VLCS scores of the twelve study subjects is depicted in FIG.3, with levels of significance. Analysis was carried out by the Friedman Test followed by Dunn's test and as shown in FIG.3, the study subjects showed a statistically significant reduction in periorbital hyperpigmentation after treatment as per Example 2.

Alternatively, the injection method was used to administer the GFC. One 5 ml dose of GFC prepared as per Example 1 was drawn into multiple 1 ml syringes. Before injection, the face was cleaned and all the make-up was removed. The periorbital 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 patient was then seated in an upright position and the injection was aimed away from the eye. The point of injection was marked with a sterile marker pen. The dose was given intra-dermally in the area of the dark circle at the seven pre-designated injection sites. The injection is also effective when given at only 3 to 5 sites. The area was disinfected thoroughly and pre-treated with a topical anaesthetic cream prior to injection. 1 ml of GFC was drawn into a 1 ml syringe with a 23-24 G needle. The needle was injected intra-dermally at a 45° angle in the orbicularis oculi muscle and 0.3 to 0.4 ml of GFC was injected at each point. A maximum volume of 2.5 ml of GFC is recommended for each dark circle. The total amount should not exceed more than 5 ml for both the dark circles of the eyes at the first sitting. One dose was found to be sufficient. A maximum of two doses can be given with an interval of 15 days between each dose.

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 POH. 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 POH. Injection of GFC at the area of dark circles causes a lightening of the skin which tends towards the normal skin colour of the study subject. 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 POH. As is evident, the dosage of GFC administered and method of administration according to an embodiment of the present invention result in significant lightening of POH. Growth factors such as TGF-β help in proliferation of fibroblasts that in-turn helps in synthesis of collagen and elastin. VEGF helps in strengthening vasculature and reduces blood cells leakage and hence the GFC proves useful for the treatment of POH by reducing inflammation, improving the skin tone and removing discolouration.

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-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 ^χ ΐθ⁶ to 5000 x lO⁶ human platelets per ml for treating periorbital hyperpigmentation, 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-dermally, sub-dermally or topically administrable growth factor concentrate derived from approximately 250 x lO⁶ to 5000 x lO⁶ human platelets per ml for treating periorbital hyperpigmentation, 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 lO⁶ human platelets per ml.

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

6. A therapeutic composition for intra-dermal, sub-dermal or topical administration for treating periorbital hyperpigmentation 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 periorbital hyperpigmentation comprising :

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 as claimed in claim 8, wherein 2.5ml of the growth factor concentrate is applied to the hyperpigmented region of the skin under each eye.

10. A method of treatment of periorbital hyperpigmentation comprising injecting a growth factor concentrate into the orbicularis oculi muscle, 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.

1 1. The method as claimed in claim 10, wherein 2.5ml of the growth factor concentrate is injected into each orbicularis oculi muscle.