WO2015049549A1

WO2015049549A1 - A method for stimulating scalp hair re-growth

Info

Publication number: WO2015049549A1
Application number: PCT/IB2013/002200
Authority: WO
Inventors: Maurizio DELNEVO
Original assignee: Beauty Thru Science Limited
Priority date: 2013-10-01
Filing date: 2013-10-01
Publication date: 2015-04-09

Abstract

The present invention relates to a method for stimulating scalp hair re-growth and for stopping hair loss in a subject in need thereof and to an extract of Aphanizomenon flos aquae (AFA) for use in said method.

Description

TITLE "A METHOD FOR STIMULATING SCALP HAIR RE-GROWTH.

FIELD OF THE INVENTION

The present invention relates to a method for stimulating scalp hair re-growth and for stopping hair loss in a subject in need thereof and to an extract of Aphanizomenon flos aquae (AFA) for use in said method.

BACKGROUND OF THE INVENTION

Male and female hair loss is frequently due to hormonal changes that are associated with stress and/or aging. In particular, genetic variations in the human androgen gene are the major determinant in the early onset of common androgenic alopecia.

Hair growth comprises cycles of various phases, i.e. the growth phase (anagen) , the regressing phase (catagen) and the resting or quiescent phase (telogen) , each of which is further divided into physiologically and morphologically distinguishable subphases.

In normal conditions, up to 90% of the hair follicles are in the anagen phase, while 10-14% are in the telogen and 1-2% in the catagen phase.

Growth cycles are controlled by a chemical signal, like an epidermal growth factor. The homeo-box protein DLX3 is a crucial regulator of hair follicle differentiation and cycling. Specifically, co-localization of Smadl/5/8 phosphorylated protein complex and DLX3 regulate the role for BMP signalling to DLx3 during hair morphogenesis in animal models.

Alopecia is the outcome of a complex process, involving a marked decrease in hair follicle size, which may develop over several years. No remedy is known in the prior art for reversing alopecia in the initial stages, or . for halting its progression.

A method to assess clinically the severity of androgenetic alopecia is provided by the Hamilton-Norwood scale for a male subject (Guarrera et al. Int. J. Trichology 2009. Jul;-Dec; 1 (2): 120-122), or of. the Ludwig scale. (Ludwig E. Sr. J. Dermatol. 1977 Sep; 97 (3) : 247-54 ) for a female subject.

Several methods have been disclosed to stop/reverse- the progression of: alopecia, but the majority of them involve surgical .. hair implantation by re-implantation of follicles from. 'the subject, ^: or topical/systemic administration of.. chemicals , which must be repeated for several months.

An object of the present invention is to provide a^' method for treatment^' and/or prevention of hair^' loss, which is effective, and ^: readily applicable to male and female subjects in need thereof.

SUMMARY OF THE INVENTION

To achieve this: object, the present - invention provides- a method for stimulating scalp hair re-growth and stopping hair loss in · a subject in need thereof,, said method comprising the steps of:

i. determining the risk factor for hereditary proneness of the subject to androgenic alopecia via a genetic screening that detects the presence or absence of a specific variation, in^' the androgen receptor gene;

ii. administering orally to the subject an extract of Aphanizomenon flos aquae (AFA) in an amount from 1100 to 1700 mg per day at least twice a day for no less than 5 days; iii. preparing a platelet-rich plasma sample via separation by centrifugation of the components in a whole blood sample from the subject or an adult adipose stem cell-rich sample by extraction of adult stem cells from a fat tissue sample from the subject, wherein the blood or fat tissue sample is obtained from the subject, within 45 to 75 minutes after the final AFA administration of step ii., in an amount dependent on the risk factor determined in step i. and on the stage of alopecia progression;

iv. mechanically stimulating the scalp of the subject by applying a scalp roller with gentle pressure; v. administering to the subject a composition comprising the platelet-rich sample of step iii. and/or the adult adipose stem cell-rich sample of step iii. via microinjections to the scalp area where hair re-growth is needed;

vi . rubbing and administering to the outer portion of the scalp a composition comprising hair growth factors (hgF) within 60 minutes from step v.;

vii. stimulating hair follicles by irradiation with a low-level laser device at a wavelength of 655 nm (+/-5%) for 5-10 minutes at least three times a week following the administration of step vi .

In another embodiment, the present invention relates to an extract of Aphanizomenon flos aquae (AFA) for use in stimulating hair re-growth and stopping hair loss in a subject in need thereof, wherein said use comprises the steps of:

i. determining the risk factor for hereditary proneness of the subject to androgenic alopecia via a genetic screening that detects the presence or absence of a specific variation in the androgen receptor gene;

ii. administering orally the extract of Aphanizomenon flos aquae (AFA) in an amount from 1100 to 1700 mg per day for no less than 5 days at least twice a day;

iii. preparing- a platelet-rich plasma. sample via separation by centrifugation of the components in a whole blood sample from the subject: or an adult adipose stem cell-rich sample by extraction of adult stem cells from a fat tissue sample from the subject, wherein the blood or fat. tissue sample is obtained from the subject, within 45 to 75 minutes after the final AFA administration of step ii., in an amount dependent, on the risk factor determined in step i. and^' on the stage of alopecia progression;

iv. mechanically stimulating the scalp of the subject by applying a scalp roller with gentle, pressure; v. administering to the scalp area of the subject where hair re-growth is needed a composition comprising the platelet-rich sample of step iii. and/or the adult adipose stem cell-rich sample of step iii . ;

vii. stimulating hair follicles by irradiation with a low-level laser device at a wavelength of 655 nm (+/-5%) for 5-10 minutes at least three times a week following the administration of step vi . .

The inventors found that subcutaneous administration to a subject of platelet-rich plasma (PRP) and/or adipose tissue adult stem cells (AT-ASCs) , in an amount that was determined on the basis of his/her specific proneness to alopecia, greatly enhances the production of new hair from follicular bulbs, that, although damaged or weak,are., not yet atrophied. The procedure according to the .invention ultimately results in arresting hair loss and promotes · actual growth of new hair, without the need for surgical procedures such as self-transplantation of hair follicles. Advantageously, in the method of the invention the PRP and AT-ASCs are obtained from the same subject to be . treated, avoiding the adverse effects commonly observed in tissue allotransplantation procedures.

FIGURE

Figure ^■ 1: modified Hamilton-Norwood scale (for. male subjects) ■ and modified Ludwig scale . .(for female subjects) .

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, in the context of the present invention percentages are referred to the weight of a single component, over the weight of the whole composition x 100.

In . one embodiment, the present invention relates to a method for stimulating scalp hair re-growth in a subject in need thereof, said method comprising the steps of:

i. determining the risk factor for hereditary proneness of the subject to androgenic alopecia via. a genetic screening that detects the presence or absence of a specific variation in the androgen receptor gene;

ii. administering orally an extract of Aphanizomenon flos aquae (AFA) in an amount from 1100 to 1700 mg per day to the subject at least twice a day for no less than 5 days; β

iii. preparing a platelet-rich plasma sample via separation by centrifugation of the- .components in a whole blood sample from the subject or an adult adipose stem cell-rich sample by extraction of adult stem cells from a fat tissue sample from the subject, wherein the blood or fat tissue sample is obtained from the subject,^, within 45 to. 75 minutes . after the final AFA administration, of step ii.-, in an amount dependent on the risk factor determined in step i. and on the stage of alopecia progression;

iv. mechanically stimulating the scalp of the 'subject by applying a scalp roller with gentle pressure;..

v. . · administering to the scalp area' of the subj.e.ct' where hair re-growth is . needed a composition comprising the platelet-rich sample of step iii.. and/or the adult adipose stem cell-rich sample of. ^' step- iii . ;

vi . ^■ rubbing and administering to the , outer portion . of the scalp a composition comprising, hair growth factors (hgF) within 60 minutes from step v. ;

v.ii. stimulating hair follicles by irradiation with alow-level laser device at a wavelength of 655 nm (+/-5%) for 5-10 minutes at least three times a week following the administration of step vi .

The method of the .invention is particularly suitable for treatment and/or prevention of male/female baldness, of hair thinning and of androgenic alopecia. Advantageously, the method of the present invention stimulates the growth of scalp stem cells and of hair follicles, contrasting excessive hair loss. It was also found that the method of the present invention enhances angiogenesis in the . scalp tissues, thereby accelerating regeneration of damaged hair follicles.

Stem cells with receptors for growth factors are present in the outer scalp tissue, particularly in the hair bulb areas. It was found that hair growth and follicle regeneration can be achieved via stimulation of these stem cells with local administration of high doses of growth factors. The method for stimulating hair re-growth in a subject in need thereof according to the present invention comprises preparing a platelet-enriched plasma sample from the same subject.

Platelets, which are contained in the blood and play a prominent role in regeneration of . bodily tissues after traumas and injuries, are naturally rich of growth factors, such as PDGF (platelet-derived growth factor) . Platelet-rich plasma extracts are used as a support treatment after surgical self-transplantation of hair follicles, to promote the growth of newly-implanted healthy follicles deriving from different body areas.

In the method of the present invention, instead, no surgical procedure is carried out but the platelet-rich preparation was surprisingly found useful to stimulate growth and trophism of follicles that are dormant or undergoing a miniaturisation process.

Because the plasma sample derives from a blood sample taken from the same subject being treated, the risk of allergic/adverse reactions is minimal.

AFA is a freshwater species of cyanobacteria . Ethanolic extracts of non-toxic AFA, particularly those collected from Upper Klamath Lake in Oregon, have been administered as food supplements. It was found that ethanol extract of AFA cellular concentrate may act to promote proliferation of human stem cell populations in vitro and in vivo (US 6,814,961).

In the method of the present invention, oral administration to the subject of an extract of Aphanizomenon flos aquae (AFA) , in an amount from 1100 to 1700 mg per day, at least twice a day for no less than 5 days was found to enhance the content of growth factors in the blood sample.

In the method of the invention, a blood sample is obtained from the subject, within 45 to 75 minutes after a dose of the AFA extract is administered to the subject. It was found that the peak of growth factor content in the blood is obtained around 1 hour after intake of the AFA extract per os. The amount of blood obtained from the subject is determined on the basis of risk factors comprising the hereditary proneness of the subject to androgenic alopecia, assessed via a genetic screening that detects the presence or absence of a specific variation in the androgen receptor gene (Ellis, J. et al J. Invest. Dermatol. 2001, 116(3), 452; Sato et al. Skin Surgery 2008, 17(2), 80) and the stage of alopecia progression, for. example according to the modified Hamilton-Norwood scale (for male subjects) or Ludwig scale (for female subjects) of Figure 1.

The blood sample is subsequently processed via centrifugation (e.g. 10 minutes at 200 RCG) to separate the platelet-rich plasma fraction from other blood components, such as leukocytes and erythrocytes in vacuum tubes, preferably containing an anti-coagulant agent sodium citrate. Suitable systems for this process include, but are not limited to, the Cytomedix Angel™ Blood Separation system.

Straight after the centrifugation, using a 10 ml syringe and an 18-gauge needle, the PRP (top layer) is drawn up/aspirated.

It was found that administration of activated platelet- rich plasma results in a long-lasting action.

Preferably, the platelet-rich plasma composition obtained in step iii. is subjected to photoactivation to stimulate the production of growth factors. The photoactivation step comprises irradiation of the sample with monochromatic visible light at different wavelengths for a total duration of about 10 minutes. Suitable methods for this step include, but are not limited to, irradiation with Adistem AdiLight-2™ Plasma Heating Device or similar.

The amount of composition comprising the . platelet-rich sample, to be administered in step v. of the method according to the invention can be determined by the practitioner on the basis of the genetic screening that detects the presence or absence of a specific variation in the androgen receptor gene and . of the .stage of alopecia progression according to the modified Hamilton- Norwood scale of Figure 1.

In case of III-IV-V class of the Hamilton-Norwood baldness pattern scheme, with high genetic alopecia risk, the practitioner can choose whether to apply a shock effect with . led-photoactivation, or to increase the number of injections as follows in the protocol.

As with reference to the modified 2013 baldness pattern evolution . (from Hamilton/Norwood, Figure 1), the practitioner can choose one of the following options:

class genetic technique inj ections treatment

risk period

2 . 1

3 low Activation 1

high Activation 1

Photo- 1/month 6 months activation

4 low Activation 1

high Activation 1

Activation 1

1 3^rd month 9 months 1 9^th month Photo- 1/month 6 months

activation

5 low Activation 1

Activation 1

1 3^rd month 9 month 1 9^th month

high Activation ^■ 1

1

Activation ^■ 1 ^'3^rd month 6 months

1 6^th month

Photo- 1/month 6 months activation

6 low Activation 1

1

Activation 1 3^rd month 6 months

1 6^th' month

high Activation ^■ 1: .

Activation ' 1 ■ '

1 3^rd month ^■ - 9, months .1 9^th month.

Activation 1

1 3^rd month ^' 9 months 1 6^th month

Photo- l./month 6 months activation

The method could also be used as preparation or follow-up to hair transplantation techniques:

• preparation: injection in the donor, and in the receiver area 30 minutes before grafts extractions;

• follow up: activated platelet-rich plasma, 1 session 3^rd. month after the procedure, 1 injection 9^th month after .

The method of the present invention further comprises mechanical stimulation of the subject's scalp by applying a scalp roller with gentle pressure, that is a moderate . pressure on the scalp that does not results in pain, discomfort or reddening/bleeding at the rolled areas.

As used herewith, the term "scalp roller" indicates a tool for stimulating microcirculation in the scalp by

..mechanical means, such as a hand-held tool comprising a handle and a rotating barrel bearing on its surface a plurality of microscopic needles (e.g. length 1.0 mm) to be applied on the scalp outer tissues.

The method of the invention further comprises administering a composition comprising the platelet-rich plasma obtained as above and/or. an adult adipose stem cell-rich sample ..obtained by extraction, of adult stem .cells^' from. a fat tissue of the subject to the subject via microinjections directly to the scalp area where hair re- growth is needed. The microinjections can be carried out at a calibrated depth of the scalp tissue with a 5 ml punch, 18-gauge needle.

In the method of the invention the activated platelet- rich plasma-comprising composition administered to the subject in step v. further comprises adult stem cells (ASC). derived from the same subject to be treated. Said adult stem cells are adipose tissue stem cells (AT-ASCs) , also known as mesenchymal ASCs (Zuk, P. A. et al. Tissue

* Engineering 2001, 7(2), 211; Zuk, P. A. ^' et al.^'Mol. Biol. .Cell. 2002, 13, 4219) . As a non-limiting example, such AT-ASC can be isolated by liposuction and/or lipoplasty and processed via the method disclosed in patent application O2010124585, the contents of which are herewith incorporated by reference. The adipose stem-rich

^■ fraction can comprise the stromal vascular fraction (SVF) of adipose tissue, SVF is a rich source of pre-^' adipocytes, mesenchymal stem cells (MSC) , endothelial progenitor cell, T cells, B cells, mast cells as well as adipose tissue macrophages and contains various different types of collagen, comprising types- 1, 3-4, 7, 14-15, 18 and 27.

Stromal Vascular Fraction (SVF) is the .product of lipoaspirate which is obtained from . liposuction of excess adipose tissue. The lipoaspirate, a. by-product ofliposuction, . contains a large population of stem cells (AT-ASCs),- which shares a number. of^: similarities with the bone marrow stromal cells, including the multilineage differentiation capacity.

.Furthermore, . the extracellular matrix, plays an important role.^' in adipocyte endocrine secretions■ and in the release of^' growth factors such as transforming growth factor beta (.TGF-β) , platelet-derived growth , factor .. ( PDGF) , and fibroblast growth factor (FGF), among -others all of which are contained in^' the SVF.17 This- is consistent with the secretions of cells in the presence ._..of an extracellular matrix. ..The SVF also contains the various proteins present in the adipose tissue .extracellular matrix of which Laminin is of interest due. to-- its. ability to help in neural regeneration. ^■.·..- ^■

The cellular composition of the SVF that - can be used in the. method^' of the present invention . ranges from:, pre- adipocytes to^' endothelial cells,..; smooth, muscle cells, pericytes, fibroblasts, and AT-ASCs. Typically, the SVF also■ contains blood cells from the capillaries supplying the fat cells. These include erythrocytes, B and T cells, macrophages, monocytes, mast cells, natural killer (NK) cells, hematopoietic stem cells-, and endothelial progenitor cells. The latter two types of cells play important roles in supporting the viability of existing blood vessels and helping create new ones respectively.- The amount of composition comprising the adipose-derived adult stem cells to be administered in step v. of . the method according to the invention can be determined by the practitioner on the basis of the genetic screening that detects the presence or absence of a specific variation in the androgen receptor gene and of the stage of alopecia progression according to the modified Hamilton-Norwood scale of Figure 1.

^•Preferably, in the method of the invention the adult stem cells are transferred using immobilized stem cell- antibody complexes. As a non—limiting example, said immobilized stem cell-antibody complexes may be obtained and transferred using the methods described in patent application WO2011034854 , the content of which are incorporated herewith by reference, comprising exposing stem cells to magnetically-sensitive antibodies to form magnetic cell-antibody complexes and transferring said complexes into the subject using: an electronically magnetizable rod.

After a single application of the composition comprising the PRP sample or the AT-ASCs of the invention according to the present invention, the concentration of growth factors was found to be 6 to 9 times higher and the CD34+ stem cells concentration was found to be 16 times higher compared to the results obtained after administration of a non-enriched plasma composition.

After rubbing the treated surface to improve release of the growth factors (which generally occurs within 60 minutes from the injection), a hair growth factor (hGF) - containing natural serum ("Evaluation of the hair growth and retention activity of two solutions on human hair explants" , 2012, Study directed by Dr E. Lati of Laboratoire Bio-EC on behalf of Pangaea laboratories Ltd.), e.g. comprising a vascular endothelial growth factor, is applied to the treated scalp area. Preferably, in the method of the invention the composition administered to the subject in step vi . comprises, human Thymosin B4 peptide and, optionally, hyaluronic acid, or the sodium salt thereof. More ^'preferably, said composition is administered by treating the scalp with a scalp roller and applying the composition topically .with gentle rubbing. ^'·^':. . . .

As a non-limiting example, said s.erum may comprise the vascular endothelial growth factor sh-Polypeptide^-9 . or CG-Thymosin-p4 (0.33% weight /weight of the total composition), sodium hyaluronate' (0; 65% weight/weight of the total composition) and .. optionally superoxide dismutase (0.80%. weight/weight of the total composition). For example, 1.0 ml of said serum may be administered each time . . ·.

Preferably, a scalp roller is .used to stimulate .. gently the scalp surface for at least '. -5 minutes before application of the composition '.comprising TB4-7 and hyaluronic acid using a dedicated infusion/air .gun. for skincare (max 50 PSI) .

As a follow-up. treatment, the method of the invention further comprises stimulating hair^' follicles by local irradiation with a low-level laser .device at a wavelength of 655 nm ( +/-5%) for 5-10 minutes :at- least three times a week.

Preferably, in addition to the above-disclosed^' steps, the method according to the present invention comprises applying a composition comprising, human Thymosin B4 peptide and hyaluronic acid, or the sodium salt thereof, once weekly for 12 weeks.

Re-growth of hair is visible after 8 weeks.

A clinical study was conducted on 38 subjects ^■ and resulted in a noticeable thickening of hair within the first 6 weeks. 90% of the subjects reported widening of hair diameter at the end of the 12-week treatment. Preferably, the method of the invention comprises, after the administration of the hgF-comprising composition of step vi . and in addition (i.e. simultaneously) to step vii., administering orally to the subject L-carnitine tartrate and/or melatonin daily for at least 9 months. More preferably, the daily amount of melatonin is from 2.5 to 4 mg (most preferably 2.9 mg) and the daily amount of L-carnitine tartrate is from 2 g to 3 g (most preferably 2.4 g) . These amounts can be administered in a single dosage or in more than one dosage throughout the day.

In addition to L-carnitine tartrate and/or melatonin, after step vi . and in addition to step vii, the method of the invention may also comprise administration of L- leucine, isoleucine, nicotinic acid, valine, zinc, biotin, selenium, iron and mixture thereof. All the above substances can be administered in a single dosage or in more than one oral form (e.g. tablet, soft or hard capsule, oral-dispersible powder, lozenge, syrup, suspension etc-. )

In another embodiment, the present invention comprises an extract of Aphanizo enon flos aquae (AFA) for use in stimulating hair re-growth and stopping hair loss in a subject in need thereof, wherein said use comprises the steps of:

i. determining the risk factor for hereditary proneness of the subject to androgenic alopecia via a genetic screening that detects the ^' presence or absence of a specific variation in the androgen receptor gene;

iv. mechanically stimulating the scalp of the subject by applying a scalp roller with gentle pressure; v. administering to the scalp area of the subject where hair re-growth is needed a composition comprising the platelet-rich sample of step iii. and/or the adult adipose stem cell-rich sample of step iii . ;

vi. rubbing and administering to the outer portion of the scalp a composition comprising hair growth factors (hgF) within 60 minutes from step vi . ;

vii.. stimulating hair follicles by irradiation with a_. low-level laser device at a wavelength of 655 nm (+/-5%) for 5-10 minutes at least three times a week.

Preferably, in the use of the invention the composition administered to the subject in step vi. comprises human Thymosin B4 peptide and, optionally, hyaluronic acid, or the sodium salt thereof.

More preferably, said composition is administered by treating the scalp with a scalp roller and applying the composition topically by gentle rubbing.

As a^■ non-limiting example, said serum may comprise the vascular endothelial growth factor sh-Polypeptide-9 or CG-Thymosin-p4 (0.33% weight/weight of the total composition), sodium hyaluronate (0.65% weight /weight of the total composition) and optionally superoxide dismutase (0.80% weight/weight of the. total composition). For example, 1.0 ml of said serum may be administered each time.

Preferably, a scalp roller is used to stimulate gently the scalp · surface for at least 5 minutes before application of the composition comprising TB4-7 and hyaluronic acid using a dedicated infusion/air gun for skincare (max 50 PSI) .

Preferably, said use further comprises photoactivating the platelet-rich plasma sample of step ii. via exposure to monochromatic light frequencies in the visible spectrum to stimulate the release of growth factors prior to the administration to the subject in step v.

Preferably, the composition administered in step v. according to the invention comprises both platelet-rich plasma and adult stem cells (ASC) derived from the same subject to be treated. More preferably, said adult stem cells in the composition administered in step v. according to the invention are adipose tissue stem cells (AT-ASCs) .

Preferably, the use of the invention comprises, after the administration of the hgF-comprising composition of step vi . and in addition (i.e. simultaneously) to step vii., administering orally to the subject L-carnitine tartrate and/or melatonin daily for at least 9 months.

More preferably, the daily amount of melatonin is from 2.5 to 4 mg (most preferably 2.9 mg) and the daily amount of L-carnitine tartrate is from 2 g to 3 g (most preferably 2.4 g) . These amounts can be administered in a single dosage or in more than one dosage throughout the day. In addition to L-carnitine tartrate and/or melatonin, after step vi . and in addition to step vii, the method of the invention may also comprise administration of L- leucine, isoleucine, nicotinic acid, valine, zinc, biotin, selenium, iron and mixture thereof. All the above substances can be administered in a single dosage or in more than one oral form (e.g. tablet, soft or hard capsule, oral-dispersible powder, lozenge, syrup, suspension etc.)

Although specific embodiments of the present invention are herewith illustrated, the invention is not limited thereto. The above descriptions are provides as exemplary of the present invention only, and should not be construed as constituting any limitation of the invention. Modifications will be obvious to the person of ordinary skill in the art that do not depart from the spirit of the invention and are intended to be included with the scope of the appended claims.

Claims

1. A method for stimulating scalp hair re-growth in a subject in need thereof, said method comprising the steps of:

ii. administering orally an extract of Aphanizomenon flos aquae (AFA) in an amount from 1100 to 1700 mg per day to the subject at least twice a day for no less than 5 days;

iii. preparing a platelet-rich plasma sample . via separation by centrifugation of the components in a whole blood sample from the subject or an adult adipose stem cell-rich sample by extraction of adult stem cells from a fat tissue sample from the subject, wherein the blood or fat tissue sample is obtained from the subject, within 45 to 75 minutes after the final AFA administration of step ii., in an amount dependent on the risk factor determined in step i. and on the stage of alopecia progression;

iv. mechanically stimulating the scalp of the subject by applying a scalp roller with gentle pressure; v. administering to the subject a composition comprising the platelet-rich sample of step iii. and/or the adult adipose stem cell-rich sample of step iii. via microinjections to the scalp area where hair re-growth is needed; vi. rubbing and administering to the outer portion of the scalp a composition comprising hair growth factors (hgF) within 60 minutes from step v.;

vii. stimulating hair follicles by irradiation with a low-level laser device at a wavelength of 655 nm ( +/- 5%) for 5-10 minutes at least three times a week following the administration of step vi .

2. The method of claim 1, wherein the composition administered in step vi . comprises human Thymosin B4 peptide and hyaluronic acid, or the sodium salt thereof.

3. The method of claim 1 or 2, further comprising photoactivating the platelet-rich plasma sample or the adult stem cell sample of step iii. via exposure to monochromatic light frequencies in the visible spectrum to stimulate the release of growth factors prior to administration to the subject in step v.

4. The method of claim 1-3, further comprising, after the administration of the hgF-comprising composition of step vi . and in addition to step vii., administering orally to the subject L-carnitine tartrate and/or melatonin daily for at least 9 months.

5. An extract of Aphanizomenon flos aquae (AFA) for use in stimulating hair re-growth and stopping hair loss in a subject in need thereof, wherein said use comprises the steps of:

ii; administering orally an extract of Aphanizomenon flos aquae (AFA) in an amount from 1100 to 1700 mg per day to the subject at least twice a day for no less than 5 days; iii. preparing a platelet-rich plasma sample via separation by centrifugation of the components in a whole blood sample from the subject or an adult adipose stem cell-rich sample by extraction of adult stem cells from a fat tissue sample from the subject, wherein the blood or fat tissue sample is obtained from the subject, within 45 to 75 minutes after the final AFA administration of step ii., in an amount dependent on the risk factor determined in step i. and on the stage of alopecia progression;

vi . rubbing and administering to the outer portion of the scalp a composition comprising hair growth factors (hgF) within 60 minutes from step vi . ; vii. stimulating hair follicles by irradiation with a low-level laser device at a wavelength of 655 nm (+/-5%) for 5-10 minutes at least three times a week .

6. The extract of AFA for use according to claim 5, wherein the composition administered in step vi . comprises human Thymosin B4 peptide and hyaluronic acid, or the sodium salt thereof.

7. The extract of AFA for use according to claim 5 or 6, further comprising photoactivating the platelet-rich plasma sample or the adult stem cell sample of step iii. via exposure to monochromatic light frequencies in the visible spectrum to stimulate the release of growth factors prior to administration to the subject in step v.

8. The extract of AFA for use according to claim 5-7, further comprising, after the administration of the hgF- comprising composition of step vi . and in addition to step vii., administering orally to the subject L- carnitine tartrate and/or melatonin daily for at least 9 months.