DMSO + Lithium Pharmaceutical Patent

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Title: Methods of modulating hair growth

United States Patent: 6,924,141

Issued: August 2, 2005

Inventors: Morgan; Bruce A. (Lexington, MA); Kishimoto; Jiro (Yokahama, JP); Burgeson; Robert (Marblehead, MA)

Assignee: The General Hospital Corporation (Boston, MA)

Appl. No.: 822722

Filed: March 30, 2001

Abstract

The invention features methods of promoting hair growth in a subject. The methods include inducing or mimicking the effects of Wnt promoted signal transduction, e.g., by increasing the level of Wnt protein or administering an agent which mimics an effect of Wnt promoted signal transduction, e.g., by administering lithium chloride. Methods of inhibiting hair growth are also provided.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery that increasing Wnt protein levels can positively regulate the ability of dermal papilla (DP) cells to promote hair growth. It was found that co-culture of Wnt expressing cells with DP cells maintains hair inductivity. In addition, it was found that agents, such as inhibitors of GSK3β kinase, e.g., lithium chloride or similar small ions, which can mimic an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, can regulate the ability of DP cells to promoter hair growth.

Accordingly, in one aspect, the invention features a method of promoting hair growth in a subject. The method includes inducing or mimicking the effects of Wnt promoted signal transduction, e.g., by increasing the level of Wnt protein or administering an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, to thereby promote hair growth.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is increased by administering an agent which increases the level of Wnt protein production and/or activity. An agent which increases the level of Wnt protein and/or which mimics an effect of Wnt promoted signal transduction can be one or more of: a Wnt polypeptide or a functional fragment or analog thereof, a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof, an agent which increases Wnt nucleic acid expression, e.g., a small molecule which binds to the promoter region of Wnt; an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Examples of agents which can mimic an effect of Wnt promoted signal transduction include: an inhibitor of GSK3β kinase, e.g., lithium chloride or similar small ions; agents which bind Frizzled (Frz) (a cell surface receptor) and mimic Wnt binding, e.g., anti-Frizzled antibodies, or other naturally or non-naturally occurring Frizzled binding ligands.

In a preferred embodiment, Wnt is increased by administering, e.g., introducing, a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof, into a particular cell, e.g., an epidermal cell or a DP cell, in the subject. The nucleotide sequence can be a genomic sequence or a cDNA sequence. The nucleotide sequence can include: a Wnt coding region; a promoter sequence, e.g., a promoter sequence from a Wnt gene or from another gene; an enhancer sequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wnt gene or from another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene or from another gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increased by increasing the level of expression of an endogenous Wnt gene, e.g., by increasing transcription of the Wnt gene. In a preferred embodiment, transcription of the Wnt gene is increased by: altering the regulatory sequences of the endogenous Wnt gene, e.g., by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the Wnt gene to be transcribed more efficiently.

In another embodiment, the method can include introducing a cell into a subject, e.g., a cell expressing Wnt. In a preferred embodiment, the cell expresses a Wnt protein, e.g., a Wnt 3, Wnt 4, or Wnt 7, or a fragment or an analog thereof. In another preferred embodiment, the cell has been genetically modified to cause the expression of Wnt, e.g., the cell has been genetically modified to express a Wnt protein, or a fragment or an analog thereof, or the cell has been genetically modified to introduce a nucleic acid sequence, e.g., a regulatory sequence, e.g., a promoter or an enhancer, that causes or increases the expression of the endogenous Wnt. In a preferred embodiment, the promoter of the endogenous Wnt gene has been replaced by another promoter, e.g., by a promoter from another gene. The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. The autologous cell is preferably from a subject characterized with hair loss. The manipulated cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a lymphocyte, a bone marrow cell, and a muscle cell. Preferably the cell is an epithelial cell, e.g., an epidermal cell, a hair follicle cell, a dermal papilla cell. The cell can be introduced into a subject to increase Wnt activity.

In a preferred embodiment, the level of Wnt, e.g., Wnt 3, Wnt 4, or Wnt 7, is increased over a sustained period of time, e.g., a period equal to or greater than 2, 10, 14, 30, 60, 90, or 180 days. E.g., a cell expressing a Wnt protein, fragment, or analog can be supplied, e.g., by any method described herein, whereby Wnt is released over a sustained period of time, e.g., a period equal to or greater than 2, 10, 14, 30, 60, 90, or 180 days.

In a preferred embodiment, the agent which increases the level of Wnt protein or mimics an effect of Wnt promoted signal transduction is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the Wnt protein level or Wnt promoted signal transduction is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair growth is promoted on: the subject’s scalp; the subject’s face, e.g., beard and/or mustache facial hair growth is promoted.

In a preferred embodiment, the subject has an insufficient amount of hair or an insufficient rate of hair growth. In a preferred embodiment, the subject suffers from genetic pattern baldness; suffers from a hormonal disorder which decreases hair growth; has received a treatment, e.g., radiation, or chemotherapy, or a drug which inhibits hair growth; or has had a surgical procedure, e.g., skin graft, which is in need of hair growth.

In another aspect, the invention features a method of inhibiting hair growth in a subject. The method includes inhibiting the level of Wnt protein or inhibiting an effect of Wnt promoted signal transduction, in the subject.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is inhibited by administering an agent which inhibits Wnt protein production levels and/or is a Wnt antagonist. An agent which inhibits Wnt can be one or more of: a Frizzled protein or Wnt binding portion thereof; a Wnt nucleic acid molecule which can bind to a cellular Wnt nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein, e.g., an antisense molecule or Wnt ribozyme; an antibody that specifically binds to Wnt protein, e.g., an antibody that disrupts Wnt’s ability to bind to its natural cellular target, e.g., disrupts Wnt’s ability to bind to Frizzled; an antibody that specifically binds to Frizzled, e.g., an antibody that disrupts Frizzled’s ability to bind to Wnt; a mutated inactive Wnt protein or fragment which binds to Frizzled but does not activate the Wnt signaling pathway; an agent which decreases Wnt gene expression, e.g., a small molecule which binds the promoter of Wnt.

In another preferred embodiment, Wnt is inhibited by decreasing the level of expression of an endogenous Wnt gene, e.g., by decreasing transcription of the Wnt gene. In a preferred embodiment, transcription of the Wnt gene can be decreased by: altering the regulatory sequences of the endogenous Wnt gene, e.g., by the addition of a negative regulatory sequence (such as a DNA-binding site for a transcriptional repressor).

In a preferred embodiment, the agent which decreases the level of Wnt protein of inhibits an effect of Wnt promoted signal transduction is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the Wnt protein level or Wnt promoted signal transduction is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair growth is inhibited on: the subject’s scalp; the subject’s face, e.g., beard and/or mustache facial hair growth or eyebrow growth is inhibited; the subject’s body hair growth is inhibited, e.g., hair growth is inhibited on the subject’s back, legs, chest, armpits.

In another aspect, the invention features a method of promoting hair growth in a subject. The method includes activating or increasing activation of the Wnt-β-catenin signaling pathway.

In a preferred embodiment, activation of the Wnt-β-catenin pathway is increased by administering an agent which increases the level of Wnt protein production and/or which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. An agent which increases the level of Wnt protein and/or which mimics an effect of Wnt promoted signal transduction can be one or more of: a Wnt polypeptide or a functional fragment or analog thereof as described herein; a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof as described herein; an agent which increases Wnt nucleic acid expression, e.g., a small molecule which binds to the promoter region of Wnt; an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Examples of agents which can mimic Wnt promoted signal transduction include inhibitors of GSK3β, e.g., lithium chloride or similar small ions, agents which bind Frizzled and mimic Wnt binding, e.g., anti-Frizzled antibodies, or other naturally or non-naturally occurring Frizzled binding ligands. In other embodiments, the level of Wnt protein can be increased by increasing the level of expression of an endogenous Wnt gene, e.g., by increasing transcription of the Wnt gene, as described herein. In another preferred embodiment, activation of the Wnt-β-catenin pathway is increased by administering: an agent which increases β-catenin protein production or activity, e.g., an agent which decreases phosphorylation of β-catenin and/or which increases β-catenin accumulation, e.g., a β-catenin polypeptide or a fragment or analog thereof, a nucleic acid sequence encoding a β-catenin polypeptide or fragments or analogs thereof; an agent which increases LEF-1 protein production and/or activity, e.g., an LEF-1 polypeptide or a fragment or analog thereof, a nucleic acid sequence encoding an LEF-1 polypeptide or fragments or analogs thereof.

In another preferred embodiment, the method can include introducing a cell, e.g., a cell which expresses and preferably secretes a protein involved in the activation of the Wnt-β-catenin signaling pathway, into a subject. In a preferred embodiment, the cell has been genetically modified to express: a Wnt protein, or a fragment or an analog thereof; a β-catenin protein, or fragment or analog thereof; an LEF-1 protein, or a fragment or analog thereof. In a preferred embodiment, the cell expresses a Wnt protein, e.g., a Wnt 3, Wnt 4, or Wnt 7, or a fragment or an analog thereof. In another preferred embodiment, the cell has been genetically modified to cause the expression of Wnt, e.g., the cell has been genetically modified to express a Wnt protein, or a fragment or an analog thereof, or the cell has been genetically modified to introduce a nucleic acid sequence, e.g., a regulatory sequence, e.g., a promoter or an enhancer, that causes or increases the expression of the endogenous Wnt. In a preferred embodiment, the promoter of the endogenous Wnt gene has been replaced by another promoter, e.g., by a promoter from another gene. The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. The autologous cell is preferably from a subject characterized with hair loss. The manipulated cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell. Preferably the cell is an epithelial cell, e.g., an epidermal cell, a hair follicle cell, a dermal papilla cell. The cell can be introduced into a subject to increase Wnt activity.

In a preferred embodiment, the level of Wnt, e.g., Wnt 3, Wnt 4, or Wnt 7, is increased over a sustained period of time, e.g., a period equal to or greater than 2, 10, 14, 30, 60, 90, or 180 days. E.g., a cell expressing a Wnt protein, fragment, or analog can be supplied, e.g., by any method described herein, whereby Wnt is released over a sustained period of time, e.g., a period equal to or greater than 2, 10, 14, 30, 60, 90, or 180 days. The cell can be introduced into a subject, e.g., to increase the level of the protein involved in activation of the Wnt-β-catenin signaling pathway.

In a preferred embodiment, the agent is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the Wnt-β-catenin signaling pathway is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair growth is promoted on: the subject’s scalp; the subject’s face, e.g., beard and/or mustache facial hair growth is promoted.

In a preferred embodiment, the subject has an insufficient amount of hair or an insufficient rate of hair growth. In a preferred embodiment, the subject suffers from genetic pattern baldness; suffers from a hormonal disorder which decreases hair growth; has received a treatment, e.g., radiation, or chemotherapy, or a drug which inhibits hair growth; or has had a surgical procedure, e.g., skin graft, which is in need of hair growth.

In another aspect, the invention features a method of inhibiting hair growth in a subject. The method includes inhibiting activation of the Wnt-β-catenin signaling pathway.

In a preferred embodiment, activation of the Wnt-β-catenin pathway is inhibited by administering an agent which decreases the level of Wnt protein production and/or decreases an effect of Wnt promoted signal transduction. An agent which inhibits the level of Wnt protein or is a Wnt antagonist can be one or more of: a Frizzled protein or Wnt binding portion thereof; a Wnt nucleic acid molecule which can bind to a cellular Wnt nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein, e.g., an antisense molecule or Wnt ribozyme; an antibody that specifically binds to Wnt protein, e.g., an antibody that disrupts Wnt’s ability to bind to its natural cellular target, e.g., disrupts Wnt’s ability to bind to a Frizzledreceptor protein; an antibody that specifically binds to Frizzled, e.g., an antibody that disrupts a Frizzled’s ability to bind to Wnt; a mutated inactive Wnt protein or fragment which binds to Frizzled but does not activate the Wnt signaling pathway; an agent which decreases Wnt gene expression, e.g., a small molecule which binds the promoter of Wnt; an agent which decreases an activity of Wnt, e.g., an agent which increases phosphorylation of β-catenin. In other embodiments, the level of Wnt protein can be inhibited by decreasing the level of expression of an endogenous Wnt gene, e.g., by decreasing transcription of the Wnt gene, as described herein. In another preferred embodiment, activation of the Wnt-β-catenin pathway is inhibited by administering: an agent which inhibits β-catenin protein production or inhibits an effect of Wnt promoted signal transduction, e.g., an agent which increases phosphorylation of β-catenin and/or which decreases β-catenin accumulation; an agent which inhibits LEF-1 protein production and/or activity.

In a preferred embodiment, the agent is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the effect on the Wnt-β-catenin signaling pathway is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair growth is inhibited on: the subject’s scalp; the subject’s face, e.g., beard and/or mustache facial hair growth or eyebrow growth is inhibited; the subject’s body hair growth is inhibited, e.g., hair growth is inhibited on the subject’s back, legs, chest, armpits.

In another aspect, the invention features a method of evaluating the status of hair growth/hair loss in a subject. The method includes evaluating, e.g., detecting, the presence or absence of a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, misexpression of the Wnt gene.

In one embodiment, the method includes evaluating whether a subject is at risk for hair loss. The method includes evaluating, e.g., detecting, a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, underexpression of the Wnt gene, to thereby determine if a subject is at risk for hair loss.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In preferred embodiment, the method includes evaluating in a sample of cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a Wnt protein. The presence of a genetic lesion is indicative of a risk of hair loss in a subject. The cell sample can be of any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a lymphocyte, a bone marrow cell, and a muscle cell.

In another preferred embodiment, the method includes evaluating in a sample of cells, e.g., a sample of epidermal cells from the hair follicle of a subject, for the expression levels of the Wnt to determine underexpression. Underexpression of Wnt is indicative of a risk of hair loss.

In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to Wnt mRNA, e.g., a labeled probe. In another preferred embodiment, expression of Wnt is evaluated with an antibody capable of binding to Wnt protein, e.g., a labeled antibody.

In another embodiment, the method includes evaluating hair growth in a subject. The method includes evaluating, e.g., detecting, absence or presence of a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, overexpression of the Wnt gene, to thereby evaluate whether hair growth is likely in a subject.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the method includes evaluating in a sample of cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a Wnt protein. The absence of a genetic lesion is indicative of a potential for hair growth. The cell sample can be of any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a lymphocyte, a bone marrow cell, and a muscle cell.

In another preferred embodiment, the method includes evaluating in a sample of cells, e.g., a sample of epidermal cells from the hair follicle of a subject, for the expression levels of Wnt to determine overexpression. Overexpression of Wnt is indicative of a potential for hair growth.

In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to Wnt mRNA, e.g., a labeled probe. In another preferred embodiment, expression of Wnt is evaluated with an antibody capable of binding to Wnt protein, e.g., a labeled antibody.

In another aspect, the invention features a method of evaluating the ability of an epidermal cell to promote hair growth or hair loss in a subject. The method includes evaluating, e.g., detecting, the presence or absence of a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, misexpression of the Wnt gene.

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In a preferred embodiment, the ability of an epidermal cell to promote hair growth or hair loss is evaluated in vitro.

In one embodiment, the method includes evaluating the ability of an epidermal cell to promote hair loss. The method includes evaluating, e.g., detecting, a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, underexpression of the Wnt gene.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt 3, most preferably, Wnt3a.

In preferred embodiment, the method includes evaluating in a sample of epidermal cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a Wnt protein. The presence of a genetic lesion is indicative of a risk of hair loss in a subject.

In another preferred embodiment, the method includes evaluating in a sample of epidermal cells, for the expression levels of the Wnt to determine underexpression. Underexpression of Wnt is indicative of a risk of hair loss.

In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to Wnt mRNA, e.g., a labeled probe. In another preferred embodiment, expression of Wnt is evaluated with an antibody capable of binding to Wnt protein, e.g., a labeled antibody.

In another embodiment, the method includes evaluating the ability of an epidermal cell to promote hair growth. The method includes evaluating, e.g., detecting, absence or presence of a genetic lesion in a Wnt gene, or evaluating, e.g., detecting, overexpression of the Wnt gene.

In a preferred embodiment, the Wnt gene or protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably, Wnt3a.

In a preferred embodiment, the method includes evaluating in a sample of epidermal cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a Wnt protein. The absence of a genetic lesion is indicative of a potential for hair growth.

In another preferred embodiment, the method includes evaluating in a sample of epidermal cells for the expression levels of Wnt to determine overexpression. Overexpression of Wnt is indicative of a potential for hair growth.

In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to Wnt mRNA, e.g., a labeled probe. In another preferred embodiment, expression of Wnt is evaluated with an antibody capable of binding to Wnt protein, e.g., a labeled antibody.

In another aspect, the invention features a method for identifying a compound capable of promoting hair growth. The method includes: contacting a cell capable of expressing a Wnt polypeptide with a test compound; and determining the level of Wnt polypeptide or nucleic acid expression, wherein a compound capable of increasing Wnt polypeptide or nucleic acid expression is indicative of a compound capable of promoting hair growth.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the compound is a Wnt fragment or analog.

In a preferred embodiment, the method further includes evaluating a control cell, e.g., an identical cell which is not treated with the compound.

In a preferred embodiment, the cell is: an epidermal cell, e.g., an epidermal cell from a hair follicle; a DP cell.

In a preferred embodiment, Wnt nucleic acid expression is evaluated using a nucleic acid probe, e.g., a labeled probe, capable of hybridizing to a Wnt nucleic acid molecule, e.g., Wnt mRNA. In a preferred embodiment, Wnt nucleic acid expression, e.g., DNA expression, is evaluated by contacting a compound with a Wnt nucleic acid molecule, e.g., a regulatory sequence of a Wnt nucleic acid molecule, and evaluating Wnt transcription, in vitro or in vivo, e.g., Wnt transcription is evaluated by determining a cell activity, e.g., using a marker gene, e.g., a lacZ gene or green fluorescence protein (GFP) gene, fused to the regulatory sequence of Wnt and following production of the marker.

In a preferred embodiment, Wnt polypeptide expression is evaluated using an anti-Wnt antibody, e.g., a labeled anti-Wnt antibody.

In another aspect, the invention features a method for identifying a compound capable of inhibiting hair growth. The method includes: contacting a cell capable of expressing a Wnt polypeptide with a test compound; and determining the level of Wnt polypeptide or nucleic acid expression in the presence and absence of the compound, wherein a compound capable of decreasing Wnt polypeptide or nucleic acid expression is indicative of a compound capable of inhibiting hair growth.

In a preferred embodiment, the Wnt polypeptide is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt nucleic acid expression is evaluated using a nucleic acid probe, e.g., a labeled probe, capable of hybridizing to a Wnt nucleic acid molecule, e.g., Wnt mRNA. In preferred embodiment, Wnt nucleic acid expression, e.g., DNA expression, is evaluated by contacting a compound with a Wnt nucleic acid molecule, e.g., a regulatory sequence of a Wnt nucleic acid molecule, and evaluating Wnt transcription, in vitro or in vivo, Wnt transcription is evaluated by determining a cell activity, e.g., using a marker gene, e.g., a lacZ gene or a GFP gene, fused to the regulatory sequence of Wnt and following production of the marker.

In a preferred embodiment, Wnt polypeptide expression is evaluated using an anti-Wnt antibody, e.g., a labeled anti-Wnt antibody.

In another aspect, the invention features a method of culturing a DP cell. For example, a human or non-human, e.g., rodent, e.g., rat or mouse, DP cell. The method includes culturing the DP cell in the presence of an increased level of Wnt, another protein involved in activating the Wnt-β-catenin signaling pathway, e.g., β-catenin and/or LEF-1, and/or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin.

In a preferred embodiment, the level of Wnt is increased over DP cells in the absence of Wnt.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, the DP cell is propagated in vitro. In a preferred embodiment, the DP cell is cultured to increase the number of DP cells.

In a preferred embodiment, a Wnt polypeptide or a functional fragment or analog thereof is added to the culture. In another preferred embodiment, an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, is added to the culture. Examples of agents which can mimic an effect of Wnt promoted signal transduction include inhibitors of GSK3β kinase such as lithium chloride or similar small ions.

In another preferred embodiment, the DP cell is cultured in the presence of a cell which expresses a Wnt polypeptide or a functional fragment or analog thereof.

In a preferred embodiment, the DP cell is obtained from a subject, cultured with an increased level of Wnt, or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, and then returned to the same or a different subject.

In a preferred embodiment, the DP cell is maintained in culture and then the cultured DP cells are returned to the same or a different subject to increase the amount of hair growth in the individual.

In another preferred embodiment, the invention features a method of providing and maintaining a dermal papilla cell graft, e.g., a DP graft for hair transplantation procedures. The method includes culturing a DP cell or DP cells in the presence of Wnt or a fragment or analog thereof, another protein involved in activating the Wnt-β-catenin signaling pathway (e.g., β-catenin and/or LEF-1) or a fragment or analog thereof, and/or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin.

In a preferred embodiment, the DP cell is propagated in vitro. In a preferred embodiment, the DP cell is propagated in vitro to increase the number of DP cells.

In a preferred embodiment, a Wnt polypeptide or a functional fragment or analog thereof is added to the culture. In another preferred embodiment, an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, is added to the culture. Examples of agents which mimic an effect of Wnt promoted signal transduction include inhibitors of GSK3β kinase such as lithium chloride or similar small ions.

In another preferred embodiment, the DP cell is cultured in the presence of a cell which expresses a Wnt polypeptide or a functional fragment or analog thereof.

In a preferred embodiment, the DP cell is obtained from a subject, cultured with an increased level of Wnt, or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, and then returned to the same or a different subject.

In another aspect, the invention features a media for culturing DP cells which includes a Wnt polypeptide or a functional fragment or analog thereof, or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Examples of agents which mimic an effect of Wnt promoted signal transduction include inhibitors of GSK3β kinase such as lithium chloride or similar small ions.

In another aspect, the invention features a method of promoting or maintaining anagen phase gene expression of DP cells. The method includes increasing the level of Wnt protein or mimicking an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, to thereby promote or maintain anagen phase gene expression in the DP cells. In another preferred embodiment, the method includes increasing activation of the Wnt-β-catenin signaling pathway, to thereby promote or maintain anagen phase gene expression in DP cells.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt level is increased or an effect of Wnt promoted signal transduction is mimicked by administering an agent which increases the level of Wnt protein production and/or which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. An agent which increases the level of Wnt protein and/or mimics an effect of Wnt promoted signal transduction can be one or more of: a Wnt polypeptide or a functional fragment or analog thereof, as described herein; a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof, as described herein; an agent which increases Wnt nucleic acid expression, e.g., a small molecule which binds to the promoter region of Wnt; an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Examples of agents which can mimic Wnt promoted signal transduction include inhibitors of GSK3β, e.g., lithium chloride or similar small ions, agents which bind Frizzled and mimic Wnt binding, e.g., anti-Frizzled antibodies, or other naturally or non-naturally occurring Frizzled binding ligands.

In a preferred embodiment, the method can be performed in vitro or in vivo. For example, the DP cells can be maintained in anagen phase in culture, and then administered to a subject, e.g., to increase hair growth. Such methods can include maintaining DP cells in culture in the presence of Wnt or an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. In one embodiment, Wnt and/or an agent which mimics an effect of Wnt promoted signal transduction, e.g., an inhibitor of GSK3β kinase, e.g., lithium chloride or similar small ions, can be added to the culture. In another embodiment, the DP cell can be co-cultured with a cell which expresses Wnt, e.g., a cell which naturally expressed Wnt or has been genetically engineered to express Wnt. DP cells maintained in anagen phase can then be used, e.g., in DP graft procedures. The DP cells can be obtained from the subject who will be receiving the DP graft (i.e., autologous cells), or can be obtained from a different subject (e.g., allogeneic or xenogeneic cells).

In a preferred embodiment, Wnt is increased by administering, e.g., introducing, a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof, into a particular cell, e.g., an epidermal cell or a DP cell, and/or into a subject. The nucleotide sequence can be a genomic sequence or a cDNA sequence. The nucleotide sequence can include: a Wnt coding region; a promoter sequence, e.g., a promoter sequence from a Wnt gene or from another gene; an enhancer sequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wnt gene or from another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene or from another gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increased by increasing the level of expression of an endogenous Wnt gene, e.g., by increasing transcription of the Wnt gene. In a preferred embodiment, transcription of the Wnt gene is increased by: altering the regulatory sequences of the endogenous Wnt gene, e.g., by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the Wnt gene to be transcribed more efficiently.

In another preferred embodiment, the method can include introducing a cell, e.g., a cell which expresses and preferably secretes a Wnt protein, into a subject. In a preferred embodiment, the cell has been genetically modified to express a Wnt protein, or a fragment or an analog thereof The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. The cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell. Preferably the cell is an epithelial cell, e.g., an epidermal cell or a DP cell. The cell can be introduced into a subject to increase the level of Wnt protein.

In a preferred embodiment, the agent which increases the level of Wnt protein and/or mimics an effect of Wnt promoted signal transduction is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the Wnt protein level is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, anagen phase gene expression is promoted or maintained in: the subject’s scalp; the subject’s face, e.g., upper lip and/or chin.

In another aspect, the invention features a method of promoting or maintaining hair inductive activity. The method includes increasing the level of Wnt protein and/or mimicking an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin, to thereby promote or maintain hair inductive activity.

In a preferred embodiment, the Wnt protein is: Wnt3, e.g., Wnt3a or Wnt 3b; Wnt 4; Wnt 7, e.g., Wnt 7a or 7b. In a particularly preferred embodiment, the Wnt protein is Wnt3, most preferably Wnt3a.

In a preferred embodiment, Wnt is increased by administering an agent which increases the level of Wnt protein production and/or activity. An agent which increases the level of Wnt protein can be one or more of: a Wnt polypeptide or a functional fragment or analog thereof; a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof; an agent which increases Wnt nucleic acid expression, e.g., a small molecule which binds to the promoter region of Wnt; an agent which mimics an effect of Wnt promoted signal transduction, e.g., inhibition of β-catenin phosphorylation, e.g., by inhibition of GSK3β kinase, or accumulation of β-catenin. Examples of agents which mimic an effect of Wnt promoted signal transduction include inhibitors of GSK3β kinase such as lithium chloride or similar small ions.

In a preferred embodiment, Wnt is increased by administering, e.g., introducing, a nucleotide sequence encoding a Wnt polypeptide or functional fragment or analog thereof, into a particular cell, e.g., an epidermal cell or a DP cell, in the subject. The nucleotide sequence can be a genomic sequence or a cDNA sequence. The nucleotide sequence can include: a Wnt coding region; a promoter sequence, e.g., a promoter sequence from a Wnt gene or from another gene; an enhancer sequence, e.g., 5′ untranslated region (UTR), e.g., a 5′ UTR from a Wnt gene or from another gene, a 3′ UTR, e.g., a 3′ UTR from a Wnt gene or from another gene; a polyadenylation site; an insulator sequence.

In another preferred embodiment, the level of Wnt protein is increased by increasing the level of expression of an endogenous Wnt gene, e.g., by increasing transcription of the Wnt gene. In a preferred embodiment, transcription of the Wnt gene is increased by: altering the regulatory sequences of the endogenous Wnt gene, e.g., by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the Wnt gene to be transcribed more efficiently.

In another preferred embodiment, the method can include introducing a cell, e.g., a cell which expresses and preferably secretes a Wnt protein, into a subject. In a preferred embodiment, the cell has been genetically modified to express a Wnt protein, or a fragment or an analog thereof. The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. The cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell. Preferably the cell is an epithelial cell, e.g., an epidermal cell or a DP cell. The cell can be introduced into a subject to increase the level of Wnt protein and/or to mimic an effect of Wnt promoted signal transduction.

In a preferred embodiment, the agent which increases the level of Wnt protein and/or mimics Wnt promoted signal transduction is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the Wnt protein level and/or the Wnt signaling pathway is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.

In a preferred embodiment, hair inductive activity is promoted or maintained on: the subject’s scalp; the subject’s face, e.g., upperlip and/or chin.

Claim 1 of 63 Claims

1. A cell culture, comprising: a dermal papilla (DP) cell, a cell culture medium, and a Wnt polypeptide or a functional fragment or analog thereof, in an amount sufficient to promote or maintain the DP cell in anagen phase.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

Source URL: http://www.pharmcast.com/Patents100/Yr2005/Aug2005/080205/6924141_Hair080205.htm

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