Link:  Pharm/Biotech Resources

Title:  Methods of inducing hair growth and coloration

United States Patent:  6,867,179

Issued:  March 15, 2005

Inventors:  Gilchrest; Barbara A. (Boston, MA); Yaar; Mina (Sharon, MA); Eller; Mark (Boston, MA)

Assignee:  Trustees of Boston University (Boston, MA)

Appl. No.:  632748

Filed:  August 4, 2000

Abstract

Methods to control, or manipulate, melanocyte and keratinocyte cell death are disclosed. In particular, a method of preventing epidermal melanocyte cell loss due to injury in a vertebrate is disclosed. Also disclosed is a method of inducing hair growth in a vertebrate, a method of inducing hair color in a vertebrate, a method of inducing skin color in a vertebrate, a method of treating baldness in an individual, and a method of treating alopecia areata in an individual.

Description of the Invention

BACKGROUND OF THE INVENTION

Normal hair follicles cycle between a growth stage (anagen), a degenerative stage (catagen), and a resting stage (telogen). The scalp hairs have a relatively long life cycle: the anagen stage ranges from two to five years, the catagen stage ranges from a few days to a few weeks, and the telogen stage is approximately three months (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, pp. 290-291; Sperling, L. C., J. Amer. Acad. Dermatology (v. 25 No. 1, Part 1), pp. 1-17 (1991)). Shorter hairs found elsewhere on the body have corresponding shorter anagen duration. The morphology of the hair and the hair follicle changes dramatically over the course of the life cycle of the hair.

During anagen, the hair follicle is highly active metabolically (Sperling, L. C., J. Amer. Acad. Dermatology (v. 25, No. 1, Part 1), p.4 (1991)). The follicle comprises a follicular (dermal) papilla at the base of the follicle; epidermal matrix cells surrounding the follicular papilla and forming the base of a hair shaft; and the hair shaft that extends upwards from the papilla through the hair canal (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993). The matrix cells are the actively growing portion of the hair (Sperling, L. C., J. Amer. Acad. Dermatology (v. 25, No. 1, Part 1), p.6 (1991)). At catagen, the matrix cells retract from the papilla, and other degenerative changes occur (Sperling, L. C., J. Amer. Acad. Dermatology (v. 25, No. 1, Part 1), pp. 13-14 (1991)). A column of epithelial cells pushes the keratinized proximal shaft of the hair upwards (Sperling, L. C., J. Amer. Acad. Dermatology (v. 25, No. 1, Part 1), p. 3 (1991)), and cell death occurs within the follicle (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, p. 291).

When the hair follicle reaches the telogen stage, the existing hair has a club-shaped proximal end, and a small bud (a remnant of the epithelial column that is found in catagen) at the base of the follicle (Sperling, L. C., J. Amer. Acad. Dermatology (v. 25, No. 1, Part 1), p. 3 (1991)). A telogen hair will not grow further (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, p. 291).

The pigmentary system that colors hair involves melanocytes located in the matrix area of the follicle, above the follicular papilla (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. 1), McGraw-Hill, Inc., 1993, p. 292). Melanin pigments produced by the melanocytes flow along dendritic processes (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, p. 292). The dendritic processes are phagocytized by the differentiating matrix cells that become part of the hair shaft; degradation of the phagocytosed material results in release of melanin granules into the cytoplasm (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, p. 671), thus pigmenting the hair.

Alterations in normal hair pigmentation or growth may be caused by age, physiologic disease conditions, or injury especially, for example, exposure to ultraviolet-irradiation. The "graying" of hair, both normal (age-associated) or abnormal, is known as canities. Graying results from a progressive decrease in pigment present in the hair shaft, caused by loss of melanocytes (Fitzpatrick, T. B., et al., eds., DERMATOLOGY IN GENERAL MEDICINE (Vol. I), McGraw-Hill, Inc., 1993, p. 671; Gilchrest, B. A., SKIN AND AGING PROCESSES, CRC Press, 1984, p. 19). A decrease in the density of hair follicles is also associated with advancing age (Gilchrest, B. A., SKIN AND AGING PROCESSES, CRC Press, 1984, p. 20).

To date, the mechanism of melanocyte and keratinocyte injury, for example, from ultraviolet exposure or the aging process, has not been determined. Thus, little is known or available regarding a mechanism to manipulate the injury process to prevent cell death and thus prevent premature baldness or graying of hair or, conversely, to promote cell death and thus, unwanted hair growth.

SUMMARY OF THE INVENTION

The present invention is based on Applicants' discovery that basal layer epidermal melanocytes and keratinocytes undergo characteristic programmed cell death in response to injury. In particular, Applicants have shown that epidermal melanocytes and keratinocytes undergo programmed cell death, or apoptosis, and that apoptosis in these cells is mediated by the p75 nerve growth factor receptor/nerve growth factor pathway (p75 NGF-R/NGF), resulting in upregulation of Bcl-2 protein. As a result of Applicants' discovery, methods are herein provided to control or manipulate (e.g., induce or maintain or inhibit or eliminate)melanocyte and keratinocyte cell death by altering the effects of apoptosis. For example, apoptosis can be inhibited using methods described herein, resulting in hair growth and coloration. Conversely, apoptosis can be promoted by methods described herein, resulting in hair loss or depigmentation.

Keratinocytes and melanocytes of the basal layer of the epidermis express high affinity (trk e.g., trk A, trk C and trk E) and low affinity (p75) NGF receptors (p75 NGF-R, or p75^NTR). p75^NTR is also referred to herein as the neurotrophin receptor and trk is referred to as the NGF-specific receptor. Neurotrophins (also referred to herein as neurotrophic factors) encompassed by the present invention include nerve growth factor (NFG), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT4/5), brain-derived neurotrophic factor (BDNF) and other neurotrophic factors that share sequence identity and structural and functional similarity to the factors mentioned above. These factors all bind to p75 NGF-R. Also encompassed by the present invention are biologically active fragments of these factors, such as the 26 kD .beta.-subunit of NGF. Biologically active fragments of the neurotrophins will bind to the p75 NGF-R. NGF, known to be produced by keratinocytes, protects cells from death when it binds to NGF receptors. In cells, this NGF effect is mediated in part by induction of the protective protein Bcl-2. Interestingly, basal epidermal keratinocytes and melanocytes express Bcl-2 protein. Specifically, as described herein, it has now been demonstrated that melanocytes expressing the p75 NGF-R can be rescued from apoptotic cell death by the occupation of the p75 NGF-R with NGF or a substance or pseudo-ligand capable of binding to the p75 NGF-R, which initiates the expression of the Bcl-2 protein. p75 contains a death domain motif and in cells that express p75, but not trk, NGF binding leads to cell death. When neurotrophins activate p75 together with receptors of the trk family, p75 evokes a survival signal. However, when p75 is activated alone, it may signal for apoptosis. As used herein, the term "activation of the receptor" means that when ligand binds to the receptor it results in initiating a pathway of cellular signaling.

Also as described herein, Applicants have now demonstrated that normal anagen hair follicles strongly express the p75 NGF-R and that p75 NGF-R expression is significantly reduced and limited to a few basal keratinocytes in telogen hair follicles.

As a result of these discoveries, methods are now available for inhibiting the process of apoptosis, or programmed cell death, in basal layer epidermal and follicular keratinocytes and melanocytes in vertebrates, specifically in humans. Thus, as a result of inhibition of apoptosis, the present invention relates to methods of inducing hair growth and coloration, and delaying hair loss and graying, as well as methods of inducing skin coloration in vertebrates. In addition, the present invention relates to methods of treating alopecia areata and baldness, as well as methods of preventing unwanted hair growth.

In one embodiment of the present invention, the invention relates to a method of preventing melanocyte and keratinocyte cell loss after injury by inhibiting apoptosis in epidermal and follicular melanocytes and keratinocytes. As described herein, Applicants have now demonstrated that p75 NGF-R mediated apoptosis is responsible for melanocyte loss after injury.

As described herein, Applicants demonstrate that p75^NTR signals for cell apoptosis when activated alone, but instead signals for cell survival when activated together with receptors of the trk family. When p75 NGF-R is present alone, binding of NGF can induce apoptosis. The binding of ligand to p75 NGF-R, where the ligand can be, for example, a neurotrophin such as NGF, a biologically active fragment of a neurotrophin, (e.g., the 26 kD .beta.-subunit of NGF) or pseudo-ligand (also referred to herein as a neurotrophin analog, or NGF analog) such as a peptide or cyclic peptide (wherein the peptide contains the sequence lysine-glycine-lysine, or lysine-glycine-alanine, in suitable three dimensional configuration for binding to p75) can inhibit the p75 NGF-R induced apoptotic pathway of cell death, resulting in the continued growth/proliferation, pigment production and pigment transfer to keratinocyte by epidermal melanocytes. Alternatively, epidermal melanocyte and keratinocyte cell loss can be prevented by upregulating the expression of Bcl-2 and other related anti-apoptotic proteins of the Bcl-2 family. Alternatively, cell loss can be prevented by downregulating the expression of the p75 NGF-R in the melanocytes and keratinocytes. Increasing the ratio of p75^NTR to trk A expression can shift the effect of NGF from one of enhancing cell survival to one of promoting apoptotic cell death in melanocytes. As further described herein, Applicants demonstrate that the apoptotic signaling pathways following activation of the p75^NTR involve sphingomyelin turnover and ceramide generation by inducing transcription of c-jun mRNA, stimulating JNK, activating caspase-3 and inducing characteristic DNA fragmentation. Signaling through the p75 receptor requires receptor aggregation, and inhibiting receptor aggregation inhibits apoptosis.

In another embodiment of the invention, the invention relates to a method of inducing hair growth in a vertebrate by upregulation of the expression of the p75 NGF-R on keratinocytes in a vertebrate, such as humans, by introducing into epidermal keratinocytes a nucleotide sequence encoding the p75 NGF-R. The p75 NGF-R gene product is expressed on the surface of the keratinocytes, and becomes available to bind to its naturally occurring ligand, NGF, or to another substance that mimics the binding activity of NGF (i.e., a pseudo-ligand). The p75 NGF-R binds its ligand, or pseudo-ligand, resulting in the expression of the protein, Bcl-2 and/or other related Bcl-2 family proteins, which protects the keratinocyte from apoptosis.

Alternatively, the upregulation of the expression of the p75 NGF-R can be accomplished by introducing into the keratinocyte a substance, such as a transcription activator protein, which initiates the transcription of the p75 NGF-R gene. It is important to remember that it is the ratio of p75 to trk that determines which signaling pathway will be activated by the neurotrophic ligand. Raising p75 level alone may enhance cell survival if the p75/trk ratio is too low.

In another embodiment of the present invention, inhibiting apoptosis in keratinocytes found in hair follicles can inhibit premature entry into catagen (hair follicle regression). Hair growth can be induced or prolonged when the keratinocyte p75 NGF-R is occupied by ligand, such as NGF, or pseudo-ligand such as a peptide or cyclic peptide containing the sequence lysine-glycine-lysine or lysine-glycine-alanine in proper conformation, which inhibits the p75 NGF-R induced apoptotic pathway of cell death, and results in the continued growth/proliferation of hair. Hair growth can also be induced or prolonged by the upregulation of the expression of the Bcl-2, and/or related proteins, protein in the keratinocytes, either by the introduction of a nucleotide sequence encoding the Bcl-2 protein or the Bcl-2 related proteins, or by the introduction of a substance that initiates transcription of the gene encoding the Bcl-2 protein, or the related protein.

In another embodiment of the present invention, the invention relates to a method of inducing hair color in a vertebrate, such as a human, by inhibiting p75 NGF-R mediated apoptosis of epidermal melanocytes.

In another embodiment of the present invention, the invention relates to a method of inducing skin color in a vertebrate, particularly a human, by inhibiting p75 NGF-R mediated apoptosis of epidermal melanocytes. As described above, Applicants have shown that peptides containing the sequence lysine-glycine-alanine (KGA) in proper conformation (e.g., a .beta.-loop conformation) specifically bind p75 NGF-R and inhibit p75 NGF-R mediated apoptosis of melanocytes.

Conversely, apoptosis can be promoted in melanocytes and keratinocytes in humans, resulting in cell death. For example, cell death may be desirable to prevent unwanted hair growth (e.g., on women's faces or forearms). This can be accomplished, for example, by blocking nerve growth factor from binding to p75 NGF-R, thereby decreasing, or completely inhibiting production of Bcl-2 protein or by promoting the activation of the p75^NTR in the absence of activating trk receptors. Thus, apoptotic cell death would be promoted.

Another embodiment of the present invention relates to a method of identifying a substance capable of inhibiting apoptosis in melanocytes or keratinocytes by determining the effect the substance has on the activation of p75 nerve growth factor receptor.

Alternatively, the method of identifying a substance capable of inhibiting apoptosis in melanocytes or keratinocytes can be accomplished by determining the effect the substance has on Bcl-2 protein expression.

Thus, as a result of Applicants' discovery of the role of p75 NGF-R induced apoptosis in epidermal and follicular melanocytes and keratinocytes, methods are now available to inhibit apoptotic cell death in epidermal and follicular melanocytes and keratinocytes, including methods of inducing or prolonging hair growth, hair coloration and skin coloration and methods of decreasing hair growth, hair coloration and skin coloration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on Applicants' finding that basal layer melanocytes and keratinocytes undergo programmed cell death, or apoptosis. Specifically, Applicants have demonstrated that melanocytes and keratinocytes of the basal layer of the epidermis and the hair follicle undergo apoptosis. Apoptosis is an active process of self-destruction that occurs in vertebrate cells. Apoptosis follows a distinct pattern of events characterized by plasma membrane blebbing, cell volume contraction, nuclear pyknosis and inter nucleosomal DNA degradation following the activation of Ca⁺ /Mg²⁺ dependent endonucleases. (Hockenberry, D. M., et al., Cell 75:241-251 (1993); Garcia, I., et al., Science 258:302-304 (1992)). Apoptosis is a highly conserved mechanism among species. Cells carry in their nuclei a genetic program for apoptosis, that can be activated upon the proper triggering, such as in response to changes in levels of hormones or growth factors in the cellular environment. (Allsopp, T. E., et al., Cell 295-307 (1993); Barinaga, M. et al., Science 259:762-763 (1992); Barinaga, M., et al., Science 263:754-755 (1994)). The "apoptotic" genes encode proteins which will induce apoptosis. However, recent evidence suggests that cells that do not undergo apoptosis express protective proteins, one of which is Bcl-2, which interact with the apoptotic proteins, sequester them and prevent their activity (Allsopp, T. E., et al., Cell 295-307 (1993)). It thus appears that a mechanism exists to protect cells from apoptosis.

To examine if UV-induced melanocyte death is apoptotic, cultures of pure human epidermal melanocytes or the human melanoma cell line MM4 (provided by Dr. U. Stierner, Goteborg, Sweden) were exposed to 5, 10 or 25 mJ/cm² UV irradiation, doses well within the physiologic UV range that reaches the basal layer of the epidermis during casual sun exposure.  Sham irradiated control cultures were handled identically but placed under a dark cloth adjacent to the UV beam. After 1-3 daily irradiations, many cells were detaching from the dish surface, while the majority of the cells in sham irradiated control cultures appeared healthy.

Total cellular DNA isolated from paired UV-irradiated cultures displayed the characteristic endonuclease-induced DNA fragmentation into multimers, the so-called DNA ladder, while DNA of sham irradiated controls was not fragmented. Duplicate UV-irradiated cultures stained with propidium iodide displayed the characteristic compaction margination and fragmentation of nuclear chromatin, as well as homogenous nuclear staining. In sham irradiated cultures, fewer than 6% of the cells stained positively with propidium iodide. In contrast, approximately 30% and 60% of cells irradiated with 10 and 25 mJ/cm² respectively were propidium iodide positive. These data strongly suggest that UV irradiation induces apoptotic death in cells of melanocytic origin.

However, melanocytes in vivo are not known to undergo apoptosis after UV-irradiation. As described herein, Applicants have demonstrated that these cells have a mechanism necessary to protect them from apoptotic cell death.

It had previously been shown that both the high affinity and low affinity nerve growth factor receptors, 140 kD and trkA and p75 NGF-R, were expressed in vitro on the surface of appropriately stimulated human melanocytes. (Peacocke, M., et al., Proc. Natl. Acad. Sci. U.S.A. 85:5282-5286 (1988); Yaar, M., et al., Clin. Res. 40:531A (1992)). It had also been shown that keratinocytes express nerve growth factor. (Yaar, M., et al., J. Cell Biol. 115:821-828 (1991); DiMarco, E., et al., J. Biol. Chem. 266:21718-21722 (1991)).

Applicants now describe herein, that nerve growth factor enhances the survival of human melanocytes after injury, for example, due to ultraviolet light exposure or growth factor deprivation.

Cultured human melanocytes were exposed to a solar simulator (5, 10, 25 mJ/cm² UVB dose) or sham irradiated and then maintained in suboptimal serum-free medium, and continuously provided with either 50 ng/ml nerve growth factor or diluent alone.  After UV irradiation, the majority of melanocytes and MM4 cells not supplemented with NGF were detaching from the dish surface.  In contrast, cultures supplemented with NGF appeared healthy.

Cell yields of melanocytes and MM4 cells irradiated with 10 mJ/cm² and supplemented with 50 ng/ml NGF were significantly higher than those of cells supplemented with diluent alone (melanocytes: 7 experiments p<0.0085; MM4 cells: 4 experiments p<0.0001, ANOVA). Furthermore, supplementation with basic fibroblast growth factor (bFGF), a major mitogen for cells of melanocytic origin (Halaban, R., et al., In Vitro Cell Devel. Biol. 23:47-52 (1987); Halaban, R., et al., J. Cell Biol. 107:1611-1619 (1988)), failed to improve MM4 cell survival after UV irradiation despite its mitogenic effect on sham irradiated cells.

To explore the mechanism of the striking response of UV irradiated cells to NGF, paired cultures were irradiated with UVB light (5, 10 or 25 mJ/cm² UVB dose), or sham irradiated, and then incubated with antibodies to the high affinity component of the NGF receptor, trk. Melanocytes in UV-treated cultures displayed more trk receptors than sham irradiated controls. Northern blot analysis checking the mRNA levels of the p75 NGF-R showed several-fold higher transcript levels in NGF-supplemented melanocytes than in diluent controls.

To determine if melanocytes undergo p75 NGF-R mediated apoptotic cell death after UV irradiation, melanocytes were exposed to UVB (10 or 25 mJ/cm²) or were sham irradiated, then maintained in suboptimal serum-free medium. Both UVB irradiation and suboptimal culture conditions, previously shown to induce p75 NGF-R expression on melanocytes, induced the DNA fragmentation patterns classic for apoptosis.

To determine whether NGF can rescue injured melanocytes from apoptosis, duplicate cultures were irradiated as described above, and maintained in medium containing 50 ng/ml NGF or diluent alone. Irradiated cultures not supplemented with NGF showed the characteristic DNA fragmentation, while cultures supplemented with NGF showed far less fragmentation. As described in "this patent", within twenty-four hours, in NGF-treated versus control melanocytes, 12% versus 30% of nuclei showed fragmentation (p less than 0.05, paired test).  Cell yields and thymidine labeling index determined daily for 19 days were higher in NGF-treated cultures (p less than 0.001), up to 6.5-fold and 10-fold, respectively.

To determine if melanocyte apoptosis is mediated by p75 NGF-R, cultures were treated as above, and then incubated in the presence of a blocking anti-human p75 NGF-R monoclonal antibody believed to act as a pseudo-ligand for the p75 NGF-R. (Anti-human p75 NGF-R monoclonal antibody courtesy of Moses V. Chao, Cornell University Medical Center, New York, N.Y.; Ross, et al., Proc. Natl. Acad. Sci. 81:6681 (1984)). Like NGF, the antibody suppressed melanocyte apoptosis in UV-irradiated cultures, while anti-rat p75 NGF-R antibody that did not bind the human p75 NGF-R had no effect.

Northern blot analysis of melanocyte RNA from donors of different ages showed that p75 NGF-R was higher in older donors, while in contrast the level for other growth factor receptors was unchanged or decreased with age, suggesting a greater vulnerability to apoptosis with aging, consistent with the clinical tendency for older persons to experience progressive hair loss.

Thus, one embodiment of the present invention relates to a method of preventing or inhibiting melanocyte cell loss after injury. The melanocytes are located in the basal epidermal layer and include melanocytes located in the skin and in hair follicles. The type of injury includes injury due to exposure to ultraviolet light, especially UVB, for example, in habitually sun-exposed skin, and injury due to the normal aging process. Injuries can also include disease conditions such as alopecia areata, telogen effluvium, and androgenic alopecia. The treatment of male-pattern baldness is also encompassed by the present invention.

More specifically, the invention relates to methods of preventing, or inhibiting, apoptosis in melanocytes and keratinocytes. As described above, Applicants have shown that apoptosis in melanocytes is mediated by the p75 NGF receptor. If the receptor is occupied, that is, if the receptor has bound an appropriate ligand, apoptosis is inhibited in the cell. Examples of appropriate ligands include neurotrophins, nerve growth factor, biologically active fragements of neurotrophins and NGF, such as the NGF 26 kD .beta.-subunit, and peptides or other small molecules that mimic the region of neurotrophins and NGF that bind to the p75^NTR, also referred to herein as p75 psuedo-ligands. Such pseudo-ligands small peptides such as the cyclic peptide, CATDIKGAEC (SEQ ID NO:9) described herein. The psuedo-ligands of the present invention bind to the p75^NTR but do not induce p75 receptor aggregation, thus do not induce apoptotic pathway signaling in the cell.

An example of an inappropriate ligand is aggregated Alzheimer's disease associated protein, .beta.-amyloid. Binding of .beta.-amyloid to the p75^NTR activates the receptor and results in apoptotic cell death of the melanocytes mediated by JNK activation.

Applicants demonstrate herein that supplementation of normal human melanocytes with .beta.-amyloid 1-40 peptide at low concentrations (.ltoreq.1 microM) leads to extensive outgrowth of dendrites, the melanocyte cell processes analogous to neurites of neurons, without decreasing cell yields. At higher concentrations of .beta.-amyloid peptide, melanocyte cell yields decrease progressively and the remaining cells appear unhealthy. Additionally, in these cultures there is focal development of plaque-like structures consisting of aggregated dying melanocytes, similar to the in vivo "senile plaques" observed in patients with AD.

Applicants further demonstrate herein that maintaining melanocytic cell cultures in .gtoreq.25 microM beta-amyloid 1-40, compared to control cultures, significantly increases the proportion of apoptotic cells and upregulates Bax protein expression approximately three fold. Recent in vivo and in vitro data suggest that the beta-amyloid induced-neuronal death exhibits classical characteristics of programmed cell death or apoptosis (Cotman, C. W. and Anderson, A. J., Mol. Neurobiol., 10:19-45 (1995); Su, J. H., et al., Neuroreport, 5:2529-2533 (1994)). The molecular pathways that regulate apoptosis in neurons have been identified in part. Evidence suggests that the product of the proto-oncogene Bcl-2 delays the onset of apoptosis in neurons that are dependent for survival on neurotrophic factors (Allsopp, T. E., et al., Cell, 73:295-307 (1993); Garcia, I., et al., Science, 258:302-304 (1992). Conversely, over expression of a 21 kD Bcl-2-associated protein, Bax, accelerates apoptotic death of cells (Oltvai, Z. N, et al., Cell, 74:609-619 (1993)).

Melanocytes express both the low affinity p75^NTR and high affinity 140 kD trk A (p140^trk A) receptors for NGF and providing NGF to melanocytes results in activation of the p140^trkA pathway, presumably through co-ordinate binding of p140^trk A and multiple p75^NTR molecules as postulated to occur in neurons exposed to NGF, that in turn activates an intracellular signal transduction pathway leading to enhanced expression of Bcl-2 and enhanced cell survival.

Applicants further demonstrate herein that beta-amyloid binds competitively to the p75^NTR. Studies previously published suggest that the specific binding site for the p75^NTR is amino acids 29-36 of the resulting NGF protein (Ulrich, A., et al., Nature 303:821-825 (1983), and that if the sequence lysine-glycine-lysine (residues 32-34 of NGF) is changed to lysine-glycine-alanine, the peptide has approximately half the affinity for the receptor as native NGF. Amino acid residues 28-30 of the beta-amyloid protein are lysine-glycine-alanine. Furthermore, computerized structure analysis of beta-amyloid reveals that these amino acids have a high probability of being in a loop turn of the protein, suggesting a high probability that this beta-amyloid peptide sequence plays a role in receptor binding.

A cyclic decapeptide was therefore synthesized by attaching two cysteine residues to the beginning and the end of the -amyloid fragment consisting of amino acids 24-31: VGSNKGAI (SEQ ID NO: 1). Cold peptide competitively inhibited ¹²⁵ I-beta-amyloid binding, with 50% inhibition occurring at 25 nM. Furthermore, beta-amyloid 200 nM reduced by cell yields by about 60% (p<0.02), but this cell loss was blocked by the peptide (200 nM). Peptide alone had no effect on cell yield. These findings indicate that apoptosis of neurons in Alzheimer's Disease results from the interaction of beta-amyloid with p75^NTR. The data also suggest that beta-amyloid-mediated death of neurons may be prevented by delivery of a synthetic peptide that blocks the beta-amyloid binding sites.

Binding of NGF to p75^NTR is mediated through amino acid residues 29-36, TDIKGKEV (SEQ ID NO: 2), that are part of the beta-hairpin loop of NGF (Ibanez, C. F., et al., Cell, 69:329-341 (1992)). If lysine (K) at position 34 is replaced by alanine (A), the resulting mutant NGF molecule still binds p75^NTR but with 50% lower affinity. Interestingly, in beta-amyloid the amino acid residues 28-30, which are present in both the 1-40 and the 25-35 beta-amyloid peptides, are KGA, a sequence that appears to permit p75^NTR binding by beta-amyloid. Computerized structure analysis of beta-amyloid suggests that the KGA residues have a high probability (>60%) of being in a loop turn, the highest probability of any portion of this 40 amino acid peptide, suggesting that this sequence constitutes a binding site for p75^NTR. Furthermore it was reported that expression of p75^NTR enhances the toxic effect of beta-amyloid on cells, possibly through binding and activation of the receptor.

Based on the above data, it is reasonable to believe that the specific three amino acid sequence lysine-glycine-alanine (KGA) in the beta-amyloid protein binds the 75 kD transmembrane neurotrophin receptor on CNS neurons, activating the programmed cell death pathway, mediated in part by an increase in intracellular Bax levels.

It is also reasonable to believe that competitively inhibiting the binding of beta-amyloid peptide blocks this aberrant receptor activation and the resulting apoptosis. For example, providing full-length NGF, or a biologically active fragment, analog, derivative, variant or mutant thereof results instead in preferential binding of p75^NTR coordinately with p140^trkA binding, leading to activation of a second signal transduction pathway that results in neuronal cell survival.

The term "biological activity" of NGF, or a fragment, derivative, analog, variant or mutant NGF, is defined herein as the activity of the NGF to specifically bind to the p75^NGF receptor. For example, an NGF mimic, or psuedo-ligand can comprise the amino acid sequence KGK or KGA, and the sequence can be in suitable three-dimensional conformation to bind to p75. Such activity can be measured by the methods described herein, or by other methods known to those skilled in the art. Another biological activity of an NGF fragment, analog, derivative, variant or mutant is the antigenic property of inducing a specific immunological response as determined using well-known laboratory techniques. For example, a biologically active NGF fragment can induce an immunological response which produces antibodies specific for the NGF (anti-NGF antibodies).

Mammalian NGF is a protein, consisting of three subunits .alpha., .beta., and .gamma., which interact to form an approximately 130 kD complex. (Ulrich, A., et al., Nature 303:821-825 (1983)). However, all known effects of NGF are mediated by the 26 kD beta-subunit through its receptor. There are two types of NGF receptors, one of a low molecular weight of approximately 75 kD, and the other of a higher molecular weight of approximately 140 kD. Both are believed necessary for the high affinity binding of NGF which is necessary for cellular response. The higher molecular weight receptor was recently found to be the protooncogene, trk, which is a member of the tyrosine kinase family. (Yaar, M., et al., J. Cell Biol., 115:821-828 (1991); Chao, M., et al., Science 232:518-521 (1986); Klein, R. S., et al., Cell 65:189-197 (1991)). NGF has been sequenced and cloned as described in Ulrich, A., et al., Nature, 303:821-825 (1983), the teachings of which are herein incorporated by reference. Thus, the entire NGF protein complex, one of its active subunits, such as the 26 kD subunit, or any biologically active fragment of NGF can be used to occupy the receptor. The biological activity of an NGF protein fragment can be determined by in vitro bioassay, for example, as described in DiMarco, E., et al., J. Biol. Chem., 266:21718-21722 (1991), the teachings of which are herein incorporated by reference.

Other substances that mimic NGF can act as a pseudo-ligand for the receptor. For example, the anti-human p75 NGF-R antibody described in Ross, et al., Proc. Natl. Acad. Sci. 81:6681 (1984) binds to p75 NGF-R and suppresses apoptosis in melanocytes. Other pseudo-ligands are KGA-containing peptides that bind p75^NTF. Examples of such pseudo-ligands are the cyclic KGA-containing hexapeptides and decapeptides described, for example, in SEQ ID NOs.4, 9 and 10. These substances include other neurotrophic factors and neurotrophins, such as NT-3, -4, and -5, with structural and functional similarity to NGF and are also capable of binding to the p75 NGF-R. (DiMarco, E., et al., J. Biol. Chem., 268:24290-24295 (1993); Yaar, M., et al., J. Invest. Derm., 100:554 (1993)). Additional substances, either protein or chemical in nature, can be produced and evaluated for their NGF-R binding ability. For example, a chemical substance can be produced that mimics the composition of NGF. This substance can be evaluated as described above for NGF activity.

Alternatively, a method of preventing epidermal melanocyte cell loss can encompass downregulating the expression of the p75 NGF-R on epidermal melanocytes. This would also result in fewer unoccupied receptor molecules and hence, suppress apoptosis and prevent melanocyte cell loss. Downregulation can be accomplished, for example, by introducing into the melanocyte a substance that inhibits or decreases the transcription of the gene encoding the p75 NGF-R. For example, an antisense oligonucleotide which is complementary to the cellular mRNA encoding the p75 NGF-R can be introduced into the melanocyte in such a manner that the antisense oligonucleotide hybridizes with the mRNA, thereby preventing translation of the mRNA into p75 NGF-R protein.

Alternatively, epidermal melanocytes can be contacted with a substance which binds to p75 nerve growth factor receptor expressed on the surface of the melanocytes. The substance, for example, can be nerve growth factor in a pharmaceutically acceptable carrier or an antibody capable of binding to p75 nerve growth factor and acting as a pseudo-ligand. Pseudo-ligands include substances that mimic nerve growth factor, such as, e.g., peptides, the KGK and KGA containing peptides described herein, organic molecules, antibodies and antibody fragments.

Pseudo-ligand antibodies which can be used in the present invention are capable of binding to p75 nerve growth factor receptor. The term antibody is intended to encompass both polyclonal and monoclonal antibodies. The preferred pseudo-ligand antibody is a monoclonal antibody reactive with a p75 nerve growth factor receptor. The term antibody is also intended to encompass mixtures of more than one antibody reactive with a p75 nerve growth factor receptor (e.g., a cocktail of different types of monoclonal antibodies reactive with a p75 nerve growth factor receptor). The term antibody is further intended to encompass whole antibodies, biologically functional fragments thereof, and chimeric antibodies comprising portions from more than one species, bifunctional antibodies, etc. Biologically functional antibody fragments which can be used are those fragments sufficient for binding of the antibody fragment to the p75 nerve growth factor receptor to occur.

The chimeric antibodies can comprise portions derived from two different species (e.g., human constant region and murine variable or binding region). The portions derived from two different species can be joined together chemically by conventional techniques or can be prepared as single contiguous proteins using genetic engineering techniques. The portions derived from two different species can also be produced by recombinant means and then joined as described above. DNA encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody can be expressed as contiguous proteins or can be produced by recombinant means and joined using techniques known to those of skill in the art.

One mechanism by which p75 induces apoptosis may involve the cellular ratio of p75^NTR to TRK in combination with the level of NGF. The abundance of NGF, the (A neurotrophin that presumptively enhances the survival of cells that express both of its receptors, decreases with age (Larkfors, L. et al. Brain Res. 1987; 427:55-60). In addition, the Applicants have found a prominent increase in the expression of the apoptotic p75^NTR with aging (Yaar, M. et al., J. Invest Deratol., 1997;108:568). These data suggest that a relative lack of NGF and/or increased levels of an apoptosis-inducing ligand such as beta-amyloid may lead to apoptotic loss of cells. Furthermore, as described herein ligand binding to the p75^NTR resulted in receptor aggregation, which led to the initiation of the apoptotic pathway. Psuedo-ligands that bind to the p75^NTR but do not aggregate the receptor, such as the cyclic peptide CATDIKGAEC (SEQ ID NO:9), can inhibit apoptosis.

The end result of p75 NGF-R and trk binding to its ligand is the expression of the protective protein, Bcl-2. Bcl-2 has been shown to prevent some classes of cell death in lymphocytes and neurons. (Veis, D. J., et a., Cell 75:229-240 (1993)). As described in "this patent", Applicants have now shown the expression of Bcl-2 by injured melanocytes after treatment with NGF. Apoptosis can be inhibited by the expression of the protective protein, Bcl-2. Thus, another method of preventing melanocyte cell loss comprises a method of upregulating expression of the Bcl-2 protein in melanocytes. This can be accomplished, for example, by inserting a nucleotide sequence encoding Bcl-2 into an expression vector capable of expressing the encoded Bcl-2 in vertebrate cells. Such an expression vector can be constructed, for example, as described in Allsopp, T. E., et al., Cell 73:295-307 (1993), the teachings of which are herein incorporated by reference. This Bcl-2 expression vector can then be introduced into melanocytes using standard laboratory techniques, such as, for example, microinjection, calcium-phosphate precipitation, or microprojectible bombardment.

Alopecia areata (AA) is a common disease of the hair follicle, affecting about 2% of new patients attending dermatology clinics in the United States and in Britain (Price, V. H., J. Invest. Dermatol., 96:685 (1991)). In alopecia areata, the hair follicle, in response to some unknown signal or injury, is suddenly precipitated into premature telogen, and then cycles in a shortened aborted cycle in which it is repeatedly arrested part way through early anagen. The follicle may remain in this arrested state but is capable of resuming normal growth after months or years. The nature of the signal or injury and the anatomical target for this abnormality are unknown.

Histologically, AA is characterized by peribulbar lymphocytic infiltrate of predominantly T helper cells (Lever, W. F. and Schaumburg-Lever, G., eds., HISTOPATHOLOGY OF THE SKIN, J.B. Lippincott Co., Philadelphia, Pa., 1990, pp. 223-224), strongly suggesting the involvement of the cellular immune system perhaps through a loss of discrimination of self and non-self antigens (Goldsmith, L. A., J. Invest. Dermatol., 96:985-1005 (1991)). Alternatively, an intrinsic abnormality in the follicular keratinocyte could be activated under the influence of internal or external triggers which eventually may lead to cellular degeneration and peribulbar inflammatory infiltrate. However, to date no specific antigen has been identified to support the autoimmune theory and no specific intrinsic difference has been reported between normal bulbar and AA keratinocytes.

As described in "this patent", indirect immunofluorescent studies were performed on biopsy material obtained from normal subjects and alopecia areata patients in an effort to detect differences in the NGF signaling system during conditions characterized by keratinocyte and melanocyte death. Results show high levels of p75 NGF-R in melanocytes and bulbar keratinocytes of the outer root sheath in the lower portion of anagen hairs, suggesting a role for p75 NGF-R in hair growth. p75 NGF-R levels were significantly reduced or p75 NGF-R was absent melanocytes and keratinocytes of telogen hairs. Furthermore, in melanocytes and keratinocytes in anagen hairs of AA patients, significantly lower levels of p75 NGF-R was also detected, indicating that decreased levels of this receptor may be involved in the pathogenesis of AA by precipitating anagen hairs into early telogen.

These findings strongly suggest that loss of the p75 NGF-R may lead to bulbar keratinocyte apoptosis and shift the hair follicle towards telogen. Decreased p75 NGF-R in anagen hair of AA patients may be the initial insult which triggers telogen effluvium in these patients.

Thus, in another embodiment of the present invention, the invention relates to a method of inducing hair growth, or maintaining hair growth in a vertebrate such as a human, by inhibiting apoptosis in keratinocytes. This is especially useful to delay or prevent hair loss in humans, for example, in male pattern baldness. Hair growth is induced by maintaining hairs in the anagen phase, and preventing the initiation of the telogen phase. As described above, p75 NGF-R levels were significantly reduced, or absent in alopecia areata patients. Thus, it is reasonable to believe that if the level of NGF-R expression on the surface of hair follicle keratinocytes is increased in the p75/trk ration is too low, the hairs are maintained in the anagen phase resulting in hair growth. Upregulating the expression of the p75 NGF-R can be accomplished by inserting a nucleotide sequence encoding the p75 NGF-R into an expression vector capable of expressing the encoded receptor protein in a vertebrate cell and introducing the receptor vector into the keratinocyte, resulting in expression of the encoded receptor. p75 NGF-R expression vectors can be constructed as described in, e.g., Rabizadeh, S., et al., Science 261:345-348 (1993); Morgenstern, J. P., et al., Nucleic Acids Res. 18:3587 (1990). This p75 NGF-R expression vector can be introduced into keratinocytes using standard laboratory techniques, such as, for example, microinjection, calcium-phosphate precipitation, or microprojectile bombardment. The cDNA sequences for human, rat and chicken p75 NGF-R are known. (Johnson, D., et al., Cell 47:545-554 (1986); Radeke, M. et al., Nature 325:593-597 (1987) and Large, T. H., et al., Neuron 2:1123-1134(1989); Huer, J. G., et al., Devl. Biol. 137:287-304 (1990), respectively, the teachings of which are incorporated by reference).

Alternatively, a substance can be introduced into epidermal keratinocytes that upregulates the expression of the p75 NGF-R, such as a transcription factor that promotes the transcription of the gene encoding the p75 NGF-R.

Based on the data presented herein, Applicants reasonably expect that p75 NGF-R/NGF mediated apoptosis also occurs in epidermal keratinocytes. Thus, the binding of p75 NGF-R and trk to ligand in epidermal keratinocytes results in the expression of the anti-apoptotic protein, Bcl-2. As described herein, p75 antagonistic cyclic peptides in a concentration of 0.01-100 micromolar significantly retard catagen development.

Another method of inducing hair growth encompassed by the present invention relates to upregulating the expression of Bcl-2 in epidermal keratinocytes. Upregulation of Bcl-2 expression can be accomplished by expressing the encoded Bcl-2 protein in keratinocytes in a similar manner as the expression of Bcl-2 protein in melanocytes as discussed above.

As discussed above, in biopsies from patients with AA, p75 NGF-R expression in keratinocytes of anagen hairs is significantly reduced or totally absent. In AA, the p75 NGF-R can be bound in vivo by a pathogenic autoantibody that precludes further binding of commercial antibodies. To pursue the possibility that reduced levels of p75 NGF-R in AA are the result of a bound autoantibody, direct immunofluorescent studies can be performed on cross section from AA patients to determine if human immunoglobulins are bound in areas known to express p75 NGF-R.

Another embodiment of the present invention relates to methods of inducing, or maintaining, hair coloration in a vertebrate comprising inhibiting apoptosis in epidermal melanocytes. Epidermal melanocytes produce melanin pigment in organelles called melanosomes and transfer the pigment to surrounding keratinocytes via extensive dendrites. Melanin pigmentation is the principal determinant of hair and skin color. inhibiting apoptosis in melanocytes results in persistently pigmented keratinocytes, or hair coloration, and thus, delays or prevents hair greying which is due to loss of hair bulb melanocytes.

Methods of therapy include administering to the individual a substance, e.g., the tripeptide KGA, or an analog thereof, in a manner which permits contact of the substance with neurons of the CNS. For example, the pentapeptide CKGAC (SEQ ID NO: 3), or an analog thereof, can be chemically synthesized by methods well-known to one of skill in the art. The cysteine residues flanking the ends of the pentapeptide can be linked, e.g., by a disulfide bond, to maintain the conformation required for binding of the peptide to the p75^NTR, thus inhibiting, or preventing apoptosis. The length of the peptide can be longer than a pentapeptide, as long as the KGA, KGK or analog peptide is maintained in a configuration suitable for binding activity. For example, as described herein, cyclic peptides have been made with the amino acid sequences and CVGSNKGAIC (SEQ ID NO: 4) these peptide compete for p75^NTR binding with beta-amyloid peptide.

Conversely, as a result of Applicants' discovery of the mechanism of apoptotic cell death in melanocytes and keratinocytes, methods are also provided that promote, apoptosis in these cells resulting in cell death. The promotion of cell death in keratinocytes may be desirable to decrease, or completely inhibit hair growth in specific areas on an individual. For example, the inhibition of facial hair growth, forearm hair growth or leg hair growth is often desirable. Thus, the methods described herein are also useful for human cosmetic purposes, e.g., for maintaining hair growth and coloration, or to remove unwanted hair.

Such inhibition of hair growth can be accomplished, for example, by the use of a blocking antibody that will block the binding of NGF to the p75 NGF-R expressed on keratinocytes. The blocking antibody (or an antibody fragment or peptide) will bind to the p75 NGF-R and thus prevent NGF from binding to the NGF-R. Thus, the NGF/p75 NGF-R mediated anti-apoptotic pathway is inhibited and cell death will be permitted, or enhanced after injury to the cells. For example, the specific area in which hair growth is to be inhibited can first be irradiated with UV light and then a composition comprising the blocking antibody can be applied (e.g., in a cream or ointment), resulting in apoptosis of injured keratinocytes and inhibition of hair growth. Alternatively, anti-trk antibodies can be used, as described herein, for activation of p75 nGF-R alone can initiate apoptosis. Furthermore, some ligands may induce apoptosis upon binding to p75 NGF-R. For example, if cell express high levels of p75 NGF-R, agregated .beta.-amyloid peptide is described herein as binding to the p75 NGF-R and inducing apoptosis.

In another embodiment of the present invention, the invention relates to in vitro methods of using the methods and compositions described herein in cell culture to, e.g., prepare epidermal melanocytes or follicular keratinocytes for transplant to vertebrates. The methods of the present invention are also useful for identifying novel substances, capable of inducing hair growth or hair coloration or inhibiting hair growth in an individual. These methods can be based on Applicants' discovery of the apoptotic mechanism of death in melanocytes and keratinocytes. An in vitro method of evaluating p75 NGF-R/NGF mediated apoptosis can use, for example, C57BL-6 mouse skin specimens with synchronized hair follicles either in telogen or anagen, as described in Paus, R., et al., Br. J. Dermatol. 122:777-784 (1990), the teachings of which are incorporated herein by reference. These skin specimens, being larger than biopsies obtained from people, and having follicles in defined portions of the growth cycle are useful to investigate the relationship between NGF/NGF-R and growth state of the hair follicle. The necessary murine probes (cDNA and antibodies) are available. For example, anti-rat p75 NGF-R antibody is available from Accurate Chemical & Scientific Company (New York) and anti-mouse NGF antibody is available from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Rat NGF cDNA is described in Maisonpierre, P. C., et al., Science 247:1446-1451 (1990) and rat p75 NGF-R cDNA is described in Radeke, M. J. et al., Nature 325:593-597 (1987). A substance to be tested for anti-apoptotic activity in melanocytes can be evaluated in this, or another a melanocyte cell culture assay. Skin specimens or melanocytes can be maintained under conditions suitable for their proliferation and then exposed to UV irradiation. After irradiation, the substance to be tested for apoptotic activity can be added to the culture system. Subsequently, the cultured cells can be evaluated to determine whether cell death has been inhibited, or decreased.

Substances identified in this method are substances that specifically alter the apoptotic mechanism in melanocytes and kerantincytes. For example, substances that mimic nerve growth factor can be tested in an assay such as described above to evaluate their activity in inhibiting apoptosis. Additionally, substances identified and evaluated by this method can be peptides, organic molecules, small organic molecules, antibodies or antibody fragments.

Substances identified using methods described herein, found to bind p75 nerve growth factor receptor, or otherwise affect p75 nerve growth factor receptor, or found to initiate Bcl-2 expression, can be used in methods to induce hair growth, hair color or skin color. These methods comprise contacting epidermal cells, including basal layer melanocytes or follicular keratinocytes, of a vertebrate with an effective amount of a substance capable of inducing hair growth, hair color or skin color by inhibiting apoptosis in melanocytes or keratinocytes. An effective amount of such an identified substance is an amount effective to significantly decrease or completely inhibit apoptotic cell death in melanocytes and keratinocytes. The decrease of inhibition of apoptosis in melanocytes and keratinocytes can be evaluated using the methods described herein.

Various delivery systems are known and can be used to administer effective amounts of substances, such as naturally-occurring ligand or pseudo-ligand for p75 nerve growth factor receptor to inhibit apoptosis in melanocytes and keratinocytes. For example, encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis, construction of a naturally-occurring or pseudo-ligand encoding nucleic acid as part of a retroviral or other vector can be used. In one embodiment, a liposome preparation can be used. The liposome preparation can be comprised of any liposomes which penetrate the stratum corneum and fuse with the cell membrane, resulting in delivery of the contents of the liposome into the cell. For example, liposomes such as those described in U.S. Pat. No. 5,077,211 of Yarosh, U.S. Pat. No. 4,621,023 of Redziniak et al. can be used.

Administration of the substances can also be, for example, by topical application to the epidermis of a vertebrate, such as a human, in a quantity sufficient to suppress apoptosis and prevent melanocyte or keratinocyte cell loss. The substance can be admixed in a pharmacological topical carrier such as a gel, an ointment, a lotion, a cream, or a shampoo and will include such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils. Other possible topical carriers include, e.g., liquid petrolatum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolauriate (5%) in water, sodium lauryl sulfate (5%) in water. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

In addition, in certain instances, it is expected that the substances can be disposed within devices placed upon, in, or under the skin. Such devices include transdermal patches, implants, and injections which release the substance in such a manner as to contact the skin or hair follicle either by passive or active release mechanisms.

The delivery vehicle can also contain perfumes, colorants, stabilizers, sunscreens, or other ingredients. The substance can be applied, for example, topically to the epidermis at regular intervals, such as once or twice daily, in a suitable vehicle and at an effective concentration. Application can also be in a vehicle which specifically targets the appropriate cells (i.e., either epidermal melanocytes or epidermal keratinocytes). For example, a membrane marker specific for melanocytes, such as melanocyte stimulating hormone (MSH), can be incorporated into a liposome containing a substance that inhibits or decreases the transcription of the gene encoding the p75 NGF-R.

An effective amount of a substance that inhibits, decreases, or promotes apoptosis can be administered to an individual using any of the above-described methods. The actual preferred amounts of a ligand to be administered will vary according to the specific ligand being utilized, the particular compositions formulated, the mode of application, and the particular situs and vertebrate being treated. The concentration of the ligand effective to suppress apoptosis and to prevent epidermal melanocyte cell loss or epidermal keratinocyte cell loss, or to promote apoptosis, in a vertebrate, such as a human, can be determined using known, conventional pharmacological protocols.

Claim 1 of 8 Claims

What is claimed is:

1. A method of maintaining or inducing hair color in a mammal, said method comprising inhibiting p75 nerve growth factor receptor-mediated apoptosis in epidermal melanocytes wherein the apoptosis is inhibited by contacting the melanocytes after injury to the skin or hair follicles, with an effective amount of a neurotrophin or a biologically active fragment thereof that binds to the p75 nerve growth factor receptor expressed on melanocytes.

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