WO2009062313A1 - Composition containing granulocyte colony stimulating factor (gcsf) for hair growth - Google Patents
Composition containing granulocyte colony stimulating factor (gcsf) for hair growth Download PDFInfo
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- WO2009062313A1 WO2009062313A1 PCT/CA2008/002019 CA2008002019W WO2009062313A1 WO 2009062313 A1 WO2009062313 A1 WO 2009062313A1 CA 2008002019 W CA2008002019 W CA 2008002019W WO 2009062313 A1 WO2009062313 A1 WO 2009062313A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
- A61K31/52—Purines, e.g. adenine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
Definitions
- 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.
- telogen resting phase
- anagen growth phase
- telogen phase regression phase
- involution of the hair follicle telogen
- bFGF basic fibroblast growth factor
- VEGF Vascular Endothelial Growth Factor
- EGF Epidermal Growth Factor
- GCSF Granulocyte Colony Stimulating Factor
- 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.
- 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.
- 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.
- the purpose of the study was to investigate the effect of growth factor GCSF on the growth of mice hair.
- GCSF was purchase from R&D System, USA (lyophilized 50 ⁇ g per vial). Test mice were injected with 50 ⁇ L of a saline solution (.9% sodium chloride in water) containing 0.1 ⁇ g GCSF (0.002 ⁇ g/ ⁇ L). Control mice were injected with 50 ⁇ L 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.
- the average hair size in the GCSF treated group on day 11 was 0.45 cm and on day 17 was 0.6 cm.
- Mixture A (for group A) Sterile saline, (.9% sodium chloride in water).
- Mixture B (for group B) 36 ⁇ g/mL of VEGF and 3 ⁇ g/mL GCSF, in saline. Prepared by mixing 50 ⁇ g VEGF in 1,260 ⁇ L saline and 40 ⁇ L stock solution G.
- Mixture C (for group C) 30 ⁇ g/mL EGF and 3 ⁇ g/mL GCSF, in saline. Prepared by mixing 80 ⁇ L stock solution E, l,180 ⁇ L saline and 40 ⁇ L stock solution G.
- Mixture D (for group D) , 36 ⁇ g/mL of VEGF, 30 ⁇ g/mL EGF and 3 ⁇ g/mL GCSF, in saline and 50 ⁇ g VEGF. Prepared by mixing 80 ⁇ L stock solution E, l,130 ⁇ L saline and 40 ⁇ L stock solution G.
- Test and control mice were injected with 50 ⁇ L one of Mixture A, B, C or D. Hair length was measured 19 days after treatment. Results
- Average hair length in the control group 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 was 10 mm.
- the average hair length in GCSF plus EGF plus VEGF 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:
- VEGF 165 amino acids
- GCSF GCSF was used at a final concentration in the mouse of 5 ⁇ g/kg per injection. Each injection therefore contained 0.1 ⁇ g. Each mouse was injected a total of six times.
- VEGF vascular endothelial growth factor
- the average control hairs were 4.5 mm long.
- the average hair length in the treated group was 6 mm.
- CMC carboxymethyl cellulose
- VEGF vascular endothelial growth factor
- the drug treated group also demonstrated thicker hair shafts.
- 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.
- 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.
- 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).
- day 0 depilation
- day 09 cyclophosphamide
- day, 10, 11, 12, 13 treatment
- days 17,18,19,and 20 treatment
- 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 ⁇ g/ ⁇ L. A ten times stock solution was prepared (.48 ⁇ g/ ⁇ L)
- GCSF GCSF was dissolved in 0.9% sodium chloride to a final concentration of 0.06 ⁇ g/ ⁇ L. A five times stock solution was made (0.3 ⁇ g/ ⁇ L)
- 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 ⁇ L CYP. Mice were then subcutaneously injected with 50 ⁇ L 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.
- 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.
- 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.
- 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.
- an extract of Tridax procumbens will be used as a topical formulation to increase the local concentration of VEGF and EGF.
- 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.
- variations that do not alter the scope of the technology are contemplated.
- 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.
- 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.
- a VEGF-stimulating factor may additionally be added to the formulation.
- 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.
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Abstract
The present technology relates to the use of Granulocyte Colony Stimulating Factor (GCSF) alone or in combination with one or more of Vascular Endothelial Growth Factor (VEGF) or Epidermal Growth Factor (EGF) in a pharmaceutical composition to inhibit alopecia and to promote hair growth. Uses of GCSF in the treatment of alopecia and in the production of a medicament for the treatment of alopecia are also provided. The technology can he used in human, veterinary and experimental medicine, more specifically in cosmetics and dermatology.
Description
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 10χ 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
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 2χ Stock CMC (8% W/V) 0.4g CMC was mixed in 5 mL 0.9% sodium chloride.
Mixture A (50 μL /mouse) 10χ VEGF stock solution 36 μL 10χ 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)
10χ 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.
Claims
1. A use of a medicament comprising Granulocyte Colony Stimulating Factor (GCSF) for the treatment of alopecia.
2. The use of claim 1 wherein the medicament further comprises Vascular Endothelial Growth Factor (VEGF).
3. The use of claim 1 or 2 wherein the medicament further comprises Epidermal Growth Factor (EGF).
4. The use of any one of claims 1 to 3 further comprising a biodegradable, polymeric carrier.
5. The use of claim 4 wherein the polymeric carrier is selected from a protein, a polysaccharide, a hydrogel, poly (D5L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and a copolymer of poly (lactic acid) and poly (caprolactone).
6. The use of claim 5 wherein the polymeric carrier one of albumin, gelatin, collagen or fibrinogen.
7. The use of claim 5 wherein the polymeric carrier is one of dextran, starch, cellulose, or carboxymethyl cellulose.
8. The use of claim 7 wherein the polymeric carrier is carboxymethylcellulose.
9. A use of Granulocyte Colony Stimulating Factor (GCSF) for the production of a medicament for the treatment of alopecia.
10. The use of claim 9 wherein the medicament further comprises Vascular Endothelial Growth Factor (VEGF).
11. The use of claim 9 or 10 wherein the medicament further comprises Epidermal Growth Factor (EGF).
12. The use of any one of claims 9 tol 1 further comprising a biodegradable, polymeric carrier.
13. The use of claim 12 wherein the polymeric carrier is selected from a protein, a polysaccharide, ahydrogel, poly (D,L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and a copolymer of poly (lactic acid) and poly (caprolactone).
14. The use of claim 13 wherein the polymeric carrier one of albumin, gelatin, collagen or fibrinogen.
15. The use of claim 13 wherein the polymeric carrier is one of dextran, starch, cellulose, or carboxymethyl cellulose.
16. The use of claim 15 wherein the polymeric carrier is carboxymethylcellulose.
17. The use of claim 15 wherein the polymeric carrier is cellulose.
18. The use of any one of claims 1 to 17 wherein the medicament is for topical application.
19. The use of any one of claims 1 to 17 wherein the medicament is for injection or implantation.
20. A composition to promote hair growth in a subject, said composition comprising: a growth factor selected from GCSF, VEGF, EGF, their derivatives and analogues, and combinations thereof; and a solvent.
21. The composition of claim 20 wherein the solvent is a sterile liquid.
14
RECTIFIED SHEET (RULE 91 .1)
22. The composition of claim 21 wherein the sterile liquid is a saline solution.
23. The composition of any one of claims 20 to 22, wherein the growth factor comprises GCSF and VEGF.
24. The composition of claim 23, further comprising EGF.
25. The composition of any one of claims 20 to 22, wherein the growth factor comprises GCSF and EGF.
26. The composition of any one of claims 20 to 25, further comprising a biodegradable polymer.
27. The composition of claim 26 wherein the biodegradable polymer is selected from a protein, a polysaccharide, a hydrogel, poly (D,L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and a copolymer of poly (lactic acid) and poly (caprolactone).
28. The composition of claim 27 wherein the biodegradable polymer is one of albumin, gelatin, collagen or fibrinogen.
29 . The composition of claim 27 wherein the biodegradable polymer is one of dextran, starch, cellulose, or carboxymethyl cellulose.
30. The composition of claim 29 wherein the biodegradable polymer is carboxymethylcellulose.
31. A composition for use in the treatment of alopecia, said composition comprising: at least one nucleic acid encoding a growth factor selected from GCSF, VEGF, EGF, their derivatives and analogues, and combinations thereof; and a solvent.
32. The composition of claim 31 wherein the solvent is a sterile liquid.
33. The composition of claim 32 wherein the sterile liquid is a saline solution.
34. The composition of any one of claims 31 to 33, wherein the nucleic acids encode GCSF and VEGF.
35. The composition of claim 34, further comprising a nucleic acid encoding EGF.
36. The composition of any one of claims 31 to 34, wherein the nucleic acids encode GCSF and EGF.
37. The composition of any one of claims 31 to 36, further comprising a biodegradable polymer.
38. The composition of claim 37 wherein the biodegradable polymer is selected from a protein, a polysaccharide, a hydrogel, poly (D,L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and a copolymer of poly (lactic acid) and poly (caprolactone).
39. The composition of any one of claims 20 to 38, further comprising a VEGF-stimulating factor.
40. A transformed cell for use in the treatment of alopecia, the cell transformed with at least one isolated nucleic acid encoding a growth factor selected from GCSF, VEGF, EGF, their derivatives and analogues, and combinations thereof.
41. The transformed cell of claim 40, wherein the nucleic acids encode GCSF and VEGF.
42. The transformed cell of claim 41, further comprising a nucleic acid encoding EGF.
43. The transformed cell of claim 40, wherein the nucleic acids encode GCSF and EGF.
44. A method of promoting hair growth in a subject, comprising: preparing a formulation comprising Granulocyte Colony Stimulating Factor (GCSF); and administering the formulation to the subject, thereby promoting hair growth.
45. The method of claim 44 wherein the medicament further comprises Vascular Endothelial Growth Factor (VEGF).
46. The method of claim 44 or 45 wherein the medicament further comprises Epidermal Growth Factor (EGF).
47. The method of any one of claims 44 to 46 further comprising a biodegradable, polymeric carrier.
48. The method of claim 47 wherein the polymeric carrier is selected from a protein, a polysaccharide, a hydrogel, poly (D,L lactide), poly (glycolide), poly (caprolactone), a copolymer of lactic acid and glycolic acid, poly(d,l-lactide-co-glycolide) and a copolymer of poly (lactic acid) and poly (caprolactone).
49. The method of claim 48 wherein the polymeric carrier one of albumin, gelatin, collagen or fibrinogen.
50. The method of claim 48 wherein the polymeric carrier is one of dextran, starch, cellulose, or carboxymethyl cellulose.
51. The method of claim 50 wherein the polymeric carrier is carboxymethylcellulose.
52. The method of any one of claims 44 to 51, wherein the medicament further comprises a VEGF-stimulating factor.
53. The method of claim 52 wherein the VEGF-stimulating factor is minoxidil or adenosine.
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PCT/CA2008/002019 WO2009062313A1 (en) | 2007-11-15 | 2008-11-13 | Composition containing granulocyte colony stimulating factor (gcsf) for hair growth |
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Cited By (2)
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US20130164252A1 (en) * | 2011-12-23 | 2013-06-27 | Saref Technologies, Inc. | Method for Hair Growth using Granulocyte-Colony Stimulating Factor |
WO2013190567A3 (en) * | 2012-06-19 | 2014-10-23 | Shome Debraj | Composition and method for an intradermal hair growth solution |
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US9827181B2 (en) | 2013-06-05 | 2017-11-28 | Industrial Technology Research Institute | Method and pharmaceutical composition for hair growth |
US9402876B2 (en) | 2013-11-27 | 2016-08-02 | Industrial Technology Research Institute | Method and pharmaceutical composition for hair growth |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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US20130164252A1 (en) * | 2011-12-23 | 2013-06-27 | Saref Technologies, Inc. | Method for Hair Growth using Granulocyte-Colony Stimulating Factor |
WO2013096723A1 (en) * | 2011-12-23 | 2013-06-27 | Saref Technologies, Inc. | A method for hair growth using granulocyte-colony stimulating factor |
US8889630B2 (en) | 2011-12-23 | 2014-11-18 | Carlos Lopez | Method for hair regrowth using Granulocyte-Colony Stimulating Factor |
US20150044163A1 (en) * | 2011-12-23 | 2015-02-12 | Carlos Lopez | A Method for Cuticle Growth using Granulocyte-Colony Stimulating Factor |
US9486501B2 (en) * | 2011-12-23 | 2016-11-08 | Carlos Lopez | Method for increasing or maintaining nail growth using granulocyte-colony stimulating factor |
WO2013190567A3 (en) * | 2012-06-19 | 2014-10-23 | Shome Debraj | Composition and method for an intradermal hair growth solution |
US9700504B2 (en) | 2012-06-19 | 2017-07-11 | Debraj Shome | Composition and method for an intradermal hair growth solution |
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