COMPOSITION CONTAINING GRANULOCYTE COLONY STIMULATING FACTOR

(GCSF) FOR HAIR GROWTH

FIELD

The present technology relates to the use of granulocyte colony stimulating factor alone or in combination with other growth factors for the treatment of alopecia.

BACKGROUND

During normal hair growth, the hair follicle undergoes a transformation from a resting phase (telogen) to a growth phase (anagen) with rapid proliferation of follicular keratinocytes and elongation and thickening of the hair shaft. Anagen phase is followed by regression phase (catagen) leading to involution of the hair follicle (telogen) which continues until a new hair shaft is generated in the existing follicle during the subsequent anagen phase. These cyclic changes involve rapid remodeling of both epithelial and dermal components of the hair follicle. In some people, the normal cycle is disrupted and hair loss occurs. This is referred to as alopecia.

It has been shown that basic fibroblast growth factor (bFGF) affects the hair follicle growth of mice in different hair cycle stages. Vascular Endothelial Growth Factor (VEGF) has also been shown to stimulate hair growth through neovascularization). Furthermore, some of the well known anti-alopecia agents such as mioxidil have been proposed to work through induction of growth factors (Lachgar et al.1998). Another growth factor that has been suggested to play important role against alopecia is Epidermal Growth Factor (EGF).

Another growth factor is Granulocyte Colony Stimulating Factor (GCSF). It belongs to the group of colony stimulating factors that regulate the differentiation and proliferation of stem cells.

SUMMARY

The present technology leads to accelerated hair re- growth and increased hair follicle size, which leads to hair thickening. Use of GCSF, VEGF, and EGF alone or in combinations thereof promotes hair growth. Further, inhibition of GCSF, VEGF, and EGF decreases hair growth and hair thickening.

FIGURES

Figure 1 shows the amino acid sequence of GCSF, in accordance with an embodiment of the technology.

Figure 2 shows the amino acid sequence of VEGF, in accordance with an embodiment of the technology.

DETAILED DESCRIPTION

The present technology is directed for use in a subject that has an insufficient amount of hair or an insufficient rate of hair growth. The subject may suffer from genetic pattern baldness, a hormonal disorder that decreases hair growth, is or has received treatment that inhibits hair growth, for example, radiation, chemotherapy or a drug that inhibits hair growth or has had a surgical procedure, such as a skin graft that is in need of hair growth. One or more of hair thickness and hair length can be increased. The technology can also be used for maintaining hair.

EXAMPLES Example 1

The purpose of the study was to investigate the effect of growth factor GCSF on the growth of mice hair.

Materials and methods

GCSF was purchase from R&D System, USA (lyophilized 50μg per vial). Test mice were injected with 50 μL of a saline solution (.9% sodium chloride in water) containing 0.1 μg GCSF (0.002 μg/μL). Control mice were injected with 50 μL of a saline solution (.9% sodium chloride in water). Each group (test and control) had 15 mice, which were tested 5 at a time. The mice were subcutaneously injected either with the GCSF solution or saline solution.

Results

Results of this study are shown below. Hair length was measured using a caliper assisted with magnifier, and an average of 20 hairs per sample were taken and reported.

The average hair size (measured as hair length) in the control mice groups at day 11 was approximately 0.3 cm and at day 17 was approximately 0.4 cm.

The average hair size in the GCSF treated group on day 11 was 0.45 cm and on day 17 was 0.6 cm.

Example 2

Materials and methods

Stock solution G O.lmg/mL GCSF in saline solution (.9% sodium chloride in water).

Stock solution E 0.5mg/mL EGF in Phosphate buffered saline.

Mixture A (for group A) Sterile saline, (.9% sodium chloride in water).

Mixture B (for group B) 36μg/mL of VEGF and 3μg/mL GCSF, in saline. Prepared by mixing 50 μg VEGF in 1,260 μL saline and 40 μL stock solution G.

Mixture C (for group C) 30μg/mL EGF and 3μg/mL GCSF, in saline. Prepared by mixing 80μL stock solution E, l,180μL saline and 40μL stock solution G.

Mixture D (for group D) , 36μg/mL of VEGF, 30μg/mL EGF and 3 μg/mL GCSF, in saline and 50μg VEGF. Prepared by mixing 80μL stock solution E, l,130μL saline and 40μL stock solution G.

Test and control mice were injected with 50 μL one of Mixture A, B, C or D. Hair length was measured 19 days after treatment.

Results

Measurement of hair length at day 19 in mm:

Average hair length in the control group (group A) was 7.8 mm and average hair length in group treated with GCSF plus VEGF (group B) was 10.8 mm. The average hair length in treated with GCSF plus EGF (group C) was 10 mm. The average hair length in GCSF plus EGF plus VEGF (group D) was 11.6 mm.

Example 3 hi order to investigate the effect of the growth factors VEGF and GCSF on the growth of mice hair, a study was designed as follows:

Materials and methods

Mice: 10 C57BL/6J 8 week old males (20 grams each).

VEGF (165 amino acids): VEGF was used at a final concentration in the mouse of 50 μg/kg per injection. Each injection therefore contained 1 μg. Each mouse was injected a total of six times.

GCSF: GCSF was used at a final concentration in the mouse of 5 μg/kg per injection. Each injection therefore contained 0.1 μg. Each mouse was injected a total of six times.

Preparation of 10x Stock solution of VEGF (0.2ug/ul):

50μg VEGF was dissolved in 0.9% sodium chloride to a final volume of 250 μL.

Preparation of 10 ^χ Stock solution of GCSF (0.02ug/ul)

5μg GCSF was dissolved in 0.9% sodium chloride to a final volume of 250 μL.

Mixture A

10x VEGF stock solution 36 μL

10x GCSF stock solution 36 μL

0.9% sodium Chloride 288 μL

Mixture B (50 μL /mouse) 0.9% sodium chloride 360 μL

Procedure

Hair was removed from the lower back area of all animals to expose approximately 2-3 cm2 of skin, using an electric hair trimmer and depilatory cream. The mice were monitored animals for 1 day to ensure there was no skin irritation. Pictures were taken of the exposed skin on all animals. Mice were subcutaneously injected with 50 μL of either Mixture A (treated) or Mixture B (control) after hair removal (Day 0) and then at days 1, 2, 7, 8, and 9. Photographs were taken on days 11, 12, 13 and 14. On day 14, hair was removed and the length measured.

Results

A summary of the results of the above study is presented in the following table.

The average control hairs were 4.5 mm long. The average hair length in the treated group was 6 mm.

Example 4

A drug formulation for slow release was used for this study. The effect of Growth factors, VEGF and GCSF in carboxymethyl cellulose (CMC) was observed on the growth of mice hair.

Materials and methods

Preparation of lOx Stock solution of VEGF (0.2ug/ul):

50μg VEGF was dissolved in 0.9% sodium chloride to a final volume of 250 μL.

Preparation of 1Ox Stock solution of GCSF (0.02ug/ul)

5μg GCSF was dissolved in 0.9% sodium chloride to a final volume of 250 μL.

Preparation of 2 ^χ Stock CMC (8% W/V) 0.4g CMC was mixed in 5 mL 0.9% sodium chloride.

Mixture A (50 μL /mouse) 10 ^χ VEGF stock solution 36 μL 10 ^χ GCSF stock solution 36 μL 2 X CMC 180 μL

0.9% sodium chloride 108 μL

Mixture B (50 μL /mouse)

0.9% sodium chloride 180 μL 2 X CMC 180 μL

Procedure

Hair was removed from the lower back area of all animals to expose approximately 2-3 cm2 of skin, using an electric hair trimmer and depilatory cream. The mice were monitored animals for 1 day to ensure there was no skin irritation. Pictures were taken of the exposed skin on all animals. Mice were subcutaneously injected with 50 μL of either Mixture A or Mixture B after hair removal (Day 0) and then at days 1, 2, 7, 8, and 9. Photographs were taken on days 11, 12, 13 and 14. On day 14, hair was removed and the length measured.

Results

Note: The drug treated group also demonstrated thicker hair shafts. By comparing the results of this example with the previous example, it can be seen that the rate of hair growth was lower when the VEGF and GCSF was administered in the absence of carboxymethyl cellulose, indicating that the carboxymethyl cellulose delayed release of the active ingredients. As would be known to one skilled in the art, other compounds and polymers could be added to VEGF and GCSF, or the other drug combinations disclosed in this application to effect slow release of the

drags.

Example 5

Angiogenesis was first studied during normal postnatal hair follicle development and during depilation-induced adult hair cycling using the chemotherapeutic drug, cyclophosphamide. C57BL/6 mice (Charles River) were sacrificed at day 0 (depilation), day 09 (cyclophosphamide), day, 10, 11, 12, 13 (treatment), then days 17,18,19,and 20 (treatment). As shown in Fig. 1 , more than a two-fold increase in hair length was observed during 19 days of the adult hair cycle.

Study design: Twenty eight C57BL/6J, male, 7 weeks old mice weighing 20 grams each were used for the study.

Cyclophosphamide monohydrate (CYP, BioChemika, 28975- Ig Sigma- Aldrich) CYP was dissolved in water to prepare a 10 times solution of 150 mg/ml).

VEGF (Human VEGF 165). VEGF was dissolved in 0.9% sodium chloride to a final concentration of 0.048 μg/ μL. A ten times stock solution was prepared (.48 μg/ μL)

GCSF GCSF was dissolved in 0.9% sodium chloride to a final concentration of 0.06 μg/μL. A five times stock solution was made (0.3 μg/ μL)

Mixture A (50 μL /mouse) 0.9% sodium chloride

Mixture B (50 μL /mouse)

10 ^χ VEGF stock solution 40 μL

0.9% sodium chloride 360 μL

Mixture C (50 μL /mouse)

10x VEGF stock solution 40 μL

0.9% sodium chloride 280 μL

5 X GCSF 80 μL

Mixture D (50 μL /mouse)

0.9% sodium chloride 320 μL

5 X GCSF 80 μL

Hair was removed from the lower back area of all animals to expose approximately 2-3 cm2 of skin, using an electric hair trimmer and depilatory cream on Day 0. The mice were monitored animals for 1 day to ensure there was no skin irritation. Pictures were taken of the exposed skin on all animals. On day 9, mice were subcutaneously injected with 100 μL CYP. Mice were then subcutaneously injected with 50 μL of either Mixture A or Mixture B at days 10, 11, 12, 13, 17, 18, 19, 20, 24 and 31 days after depilation. Note that the injection site was moved 0.25 cm each day. Photographs were taken on days 8, 9, 12, 13 and days thereafter until there was 75% restoration of the hair. On day 31 , hair was removed and the length measured.

Results

Twenty hairs from each mouse were measured and the hairs for each treatment pooled. The average length is summarized in the following table:

The results of this study demonstrated that cyclophosphamide (CYP) caused severe alopecia and delayed hair growth. After 31 days the animal hairs were very short at an average length of 3.5 mm (CYP + Saline). However, if the animals were treated with VEGF or GCSF or combination of both after the alopecia then the hair grew rapidly. The animals treated with VEGF alone showed an average hair length of 5.4 mm (CYP + VEGF), and the animals treated with GCSF showed an average hair length of 6 mm (CYP + GCSF). The best results were seen when the

animals received both GCSF and VEGF. The average hair length was 7 mm (CYP + GCSF). Hair on the animals that were not treated with CYP grew normally and attained an average hair length of 7.5 mm (No CYP). hi conclusion, both VEGF and GCSF had the potential to restore the cyclophosphamide-induced alopecia rapidly.

Example 6

The relationship between hair growth enhancement observed during the growth phase and increases in the size of hair follicles or in the thickness of the overlying epidermis, the dermis and the subcutis was examined. It was found that the thickness of interfollicular epidermis significantly increased from day 10 to reach a maximum thickness on day 25 (Fig. 2). The temporal coincidence of the cyclic changes in follicle, dermal and subcutaneous thickness, and perifollicular vascularization suggested that the stimulus leading to hair growth was associated with increased in stem cell-induced angiogenesis and growth factors from the hair follicle itself or from the surrounding dermis and subcutis keratinocytes.

Example 7

One or more of GCSF, EGF and VEGF levels will be increased by administering to a patient, at least one of the nucleotide sequences encoding GCSF, EGF or VEGF polypeptides or functional fragment or analog thereof. GCSF, EGF, VEGF levels or levels of combinations thereof will be increased in a particular cell, such as a keratinocyte, by upregulating expression in a cell specific manner, or by introducing a transgene. Alternatively, GCSF, EGF or VEGF levels or levels of combinations thereof will be increased in a location-specific manner, that location being the scalp, beard, chest or back of a patient in need of treatment, by upregulating expression in a location-specific manner, or by introducing a transgene. Modes of administration will be topical, systemic, oral, nasal, implantation or injection dermally or subcutaneously. A surfactant or an agent which increases permeability in the skin will be used to assist in topical administration. Detergents such as SDS, DMSO, bile salts, and fusidic acid derivatives may be used for this purpose. Injections will be delivered as sterile formulations, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation will be vacuum drying and freeze-drying. Specific transduction of the protein in the target cells will be

controlled by the specificity of transfection provided by the gene delivery vehicle. Cell or tissue specific expression will be controlled by regulatory sequences. Alternatively, genetically engineered cells will be introduced. The cells can be any cell type, but will preferably be fibroblasts, a keratinocytes, epithelial cells, endothelial cells, glial cells or precursors of these somatic cell types. Cells maybe obtained from a donor of the same species or another species. The cells can be administered in a gel or a biocompatible mesh.

Example 8

One or more of GCSF, EGF and VEGF protein levels will be increased by administering to a patient, one or more of GCSF, EGF, VEGF, combinations thereof or functional fragments or analogs thereof. Modes of administration will be topical, systemic, oral, nasal, implantation or injection dermally or subcutaneously. Injections will be delivered as sterile formulations. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation will be vacuum drying and freeze-drying. A surfactant or an agent which increases permeability in the skin will be used to assist in topical administration. Detergents such as SDS, DMSO, bile salts, and fusidic acid derivatives may be used for this purpose. The proteins or a functional fragment or analog thereof.may be delivered in a gel, cream, or liquid. In an exemplary case, an extract of Tridax procumbens will be used as a topical formulation to increase the local concentration of VEGF and EGF.

Example 9

One or more of GCSF, EGF and VEGF levels will be increased by administering to a patient an agent or molecule that increases GCSF, EGF, VEGF gene expression or gene expression of combinations thereof. The molecule will be a stem cell stimulating factor, an FLT3 ligand, a cytokine,or an interleukin-1. Modes of administration will be topical, systemic, oral, nasal, implantation or injection dermally or subcutaneously. Injections will be delivered as sterile formulations. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation will be vacuum drying and freeze-drying. A surfactant or an agent which increases permeability in the skin will be used to assist in topical administration. Detergents such as SDS, DMSO, bile salts, and fusidic acid derivatives may be used for this purpose. The agent or molecule may be delivered in a gel, cream, or liquid.

The foregoing is a description of an embodiment of the technology. As would be known to one skilled in the art, variations that do not alter the scope of the technology are contemplated. For example, it is contemplated that the growth factors, GCSF, VEGF, EGF, could be used alone, or in any combination two or three of GCSF, VEGF, EGF. A wide range of solvents of the growth factors are contemplated, including a sterile saline solution. Any biodegradable polymer that is biocompatible could be used with the growth factor to effect slow release, for example, but not limited to a protein such as albumin, gelatin, or fibrinogen, a polysaccharide such as dextran, starch, cellulose, or carboxymethyl, poly (D,L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and copolymer of poly (lactic acid) and poly (caprolactone). The GCSF, VEGF, EGF, and combinations thereof may be provided as a gene or genes expressing the GCSF, VEGF, EGF and combinations thereof. Further, a VEGF-stimulating factor may additionally be added to the formulation. In this regard, adenosine or minoxidil are contemplated, but as would be known to one skilled in the art, any VEGF-stimulating factor maybe added singly, or in combination with other VEGF-stimulating factors.