NOVEL COMPOSITIONS AND USES THEREOF

Abstract

The present invention provides a composition for stimulating hair growth in a mammal comprising a modified osteopontin polypeptide in which an RGD domain is inactivated; and a pharmaceutically acceptable and/or cosmetically acceptable excipient, carrier or diluent. The invention further provides methods of stimulating hair growth in a mammal.

Claims

1-111. (canceled)

112. A method for stimulating hair growth in a mammal comprising administering to the mammal an effective amount of a composition comprising: (a) a modified osteopontin polypeptide comprising of the amino acid sequence of SEQ ID NO: 63 or a fragment or variant thereof; and (b) a pharmaceutically acceptable and/or cosmetically acceptable excipient, carrier or diluent.

113. The method according to claim 112 consisting of a fragment of the amino acid sequence of SEQ ID NO: 63.

114. The method according to claim 112 wherein the modified polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 3 TABLE-US-00028 SEQIDNO:3 MRIAVICFCLLGITCAIPVKQADSGSSEEKQLYNKYPDAVATWLNPDPSQ KQNLLAPQTLPSKSNESHDHMDDMDDEDDDDHVDSQDSIDSNDSDDVDDT DDSHQSDESHHSDESDELVTDFPTDLPATEVFTPVVPTVDTYDGDISVVY GLRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQDLNAPS DWDSRGKDSYETSQLDDQSAETHSHKQSRLYKRKANDESNEHSDVIDSQE LSKVSREFHSHEFHSHEDMLVVDPKSKEEDKHLKFRISHELDSASSEVN.

115. The method according to claim 112 wherein said administering stimulates existing hair follicles in the mammal.

116. The method according to claim 112 wherein said administering induces the growth of new hair follicles, or stem cells for producing new hair follicles in the mammal.

117. The method according to claim 112 wherein said administering treats or prevents a disease or condition associated with hair loss in the mammal.

118. The method according to claim 112 wherein the administering treats or prevents alopecia in the mammal.

119. The method according to claim 118 wherein the alopecia is associated with the loss of anagen hairs.

120. The method according to claim 118, wherein the alopecia is selected from the group consisting of: (a) androgenic alopecia (also known as androgenetic alopecia, alopecia androgenetica, male pattern baldness or female pattern baldness); (b) traction alopecia; (c) anagen effluvium; (d) telogen effluvium; (e) alopecia areata; (f) alopecia totalis; (g) alopecia universalis; (h) alopecia barbae; (i) alopecia mucinosa; (j) alopecia neoplastica; (k) cicatricial alopecia; and (l) scarring alopecia.

121. The method according to claim 118 wherein the alopecia is androgenic alopecia.

122. The method according to claim 118 wherein the alopecia is anagen effluvium.

123. The method according to claim 112 wherein the mammal has hair loss induced by radiotherapy and/or chemotherapy agents.

124. The method according to claim 112 wherein the mammal is a human.

Description

[0212] Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

[0213] FIG. 1 shows a schematic representation of the sampling area of skin on the mice.

[0214] FIG. 2 is a representative photograph of a cross section of skin from a mouse 48 hours after being treated with a modified osteopontin polypeptide. Follicles within the epidermis are indicated by the arrow. The linear density of the follicles was 18.3 follicles per mm (598 follicles in 32.65 mm of epidermis).

[0215] FIG. 3 is a representative photograph of a cross section of skin from a mouse 48 hours after being treated with a negative control composition. Follicles within the epidermis are indicated by the arrow. The linear density of the follicles was 11.7 follicles per mm (379 follicles in 32.50 mm of epidermis).

[0216] FIG. 4 shows representative photographs of (a) a control-treated animal on day 14 having a hair growth score of 0, (b) an animal treated with exemplary full length polypeptide Cx (corresponding to SEQ ID NO: 1 but without the initial sixteen amino acid signal peptide, 60 nM) on day 14 having a hair growth score of 1.5 (c) an animal treated with exemplary short peptide FOL-004 (SEQ ID NO: 5, 60 nM) on day 5 having a hair growth score of 2.5 and (d) an animal treated with exemplary short peptide FOL-005 (SEQ ID NO: 5, 63 nM) on day 5 having a hair growth score of 2.0.

[0217] FIG. 5 shows the effect of exemplary full length polypeptide Cx and polypeptides SEQ ID NOs: 5 and 63 of the invention on hair growth.

[0218] FIG. 6 shows the effect of exemplary polypeptide SEQ ID NO: 5 on hair growth, as compared to the corresponding polypeptide SEQ ID NO: 121 from wildtype mouse osteopontin.

EXAMPLE A

In Vivo Study of Hair Growth Effect of Mutated Mouse Osteopontin in Mice

[0219] Materials and Methods

[0220] Creation and Production of Test Polypeptide

[0221] The modified osteopontin polypeptide sequence SEQ ID NO:1 was cloned into a pCEP4 expression vector by the use of Xhol and BamHI restriction enzyme sites. The pCEP4 expression vector contains an ampicillin resistance gene for expression in bacteria and a hygromycin resistance gene for expression in eukaryotic EBNA cells. The polypeptide containing vector were transformed into competent XL-10 bacteria cells, multiplied and isolated with Qiagen midi prep kit. Isolated vectors were then transfected into human EBNA cells by the use of Fugene transfection reagent according to manufacture protocol (Invitrogen).

[0222] Isolation of Test Polypeptide

[0223] Medium from EBNA cell culture containing the produced polypeptide were collected after three to four days of culture. Polypeptides produced by pCEP4 plasmid contain an inserted His-tag, which is used to facilitate the isolation with Ni-sepharose gel chromatography (Invitrogen). Collected medium was diluted with binding buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8), Ni-sepharose suspension was added before incubation on shaker overnight at 4 C. The Ni-sepharose gel was pelleted by centrifugation at 1000 g for seven minutes and poured into a BioRad disposable mini column. Unbound proteins were removed with binding buffer followed by washing buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 6.0). Polypeptides were eluated from the column with 500mM imidazole.

[0224] General Study Design

[0225] The study consisted of four experimental groups each containing five male wtC57BL/6N mice.

[0226] On day 1 the necks of all participating mice were shaved carefully in squares of approx. 1.51.0 cm and small remaining hairpieces were removed. On the following day (day 0), each animal received four intracutaneous injections of 25 l each at separate locations (each approx. 0.50.5 cm) within the shaved rectangle (see FIG. 1).

[0227] Each animal received two injections of 25 l of a composition comprising an exemplary modified osteopontin polypeptide of SEQ ID NO:1 (Test polypeptide or Cx, 60 nmol/l in PBS) and two negative control injections of 25 l PBS (according to the scheme outlined in Table 1).

TABLE-US-00010 TABLE 1 Study Design Intracutaneous Application Number Time schedule after of Group Volume application animals 1 Test 25 l Necropsy after 24 h (day 1) 5 polypeptide + PBS 2 Test 25 l Necropsy after 48 h (day 2) 5 polypeptide + PBS 3 Test 25 l Necropsy after 96 h (day 4) 5 polypeptide + PBS 4 Test 25 l Necropsy after 336 h (day 14) 5 polypeptide + PBS

[0228] Animals from Treatment Groups 1 to 4 were sacrificed 24 h (Group 1), 48 h (Group 2), 96 h (Group 3) and 336 h (14 days, Group 4) after compound application, respectively.

[0229] In all cases, the marked skin areas in the neck were removed and processed as follows: One of the polypeptide-treated skin samples and one of the PBS-treated control skin samples were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. The two other skin samples (polypeptide-treated and PBS-treated) were snap-frozen in liquid nitrogen and stored appropriately at 80 C.

[0230] A Hematoxylin-staining of the paraffin-embedded sections was performed.

[0231] Animals

TABLE-US-00011 Rationale Accepted test system for the purpose of the study Strain male wtC57BL/6N Source Charles River GmbH Sandhofer Weg 7 D 97633 Sulzfeld Total number of animals 20 Age at delivery 5-6 weeks Body weight and range approx. 30 g (at acclimatisation) Identification Labeling by ear mark Acclimatisation February, 17.sup.th to March, 1.sup.st 2011

[0232] Husbandry

TABLE-US-00012 Conditions Optimum hygienic conditions, air-conditioned with 10-15 air changes per hour, and continually monitored environment with target ranges for temperature 22 3 C. and for relative humidity 30- 70%, 12 hours artificial fluorescent light/12 hours dark. Accommodation max. 3 animals per cage Diet M-Zucht ssniff Spezialditen GmbH Ferdinand Gabriel Weg 16 D-59494 Soest Water Community tap water (autoclaved)

[0233] Test Polypeptide

TABLE-US-00013 Identification Modified osteopontin polypeptide of SEQ ID NO: 1 Storage conditions stored in Hepes buffer at 4 C. Safety precautions prevent ingestion, inhaling, wear gloves and skin mask, wash with soap and water after skin contact; no special precautions Sample preparation delivered stock solution was diluted 15x with PBS to obtain the 60 nM working solution before injection

[0234] Vehicle

TABLE-US-00014 Identity PBS pH 7.4

[0235] Treatment

TABLE-US-00015 Route of administration: intracutaneously Frequency of administration single application on day 0 Dose volume 25 l

[0236] Observations

[0237] The following parameters were recorded:

TABLE-US-00016 Viability/Mortality daily Clinical signs daily

[0238] Protocol for Hematoxylin-Staining of Paraffin-Embedded Sections of Group 1

[0239] 1. Xylen bath 5 min

[0240] 2. Xylen bath 5 min

[0241] 3. EtOH 100% bath 5 min

[0242] 4. EtOH 95% bath 5 min

[0243] 5. EtOH 70% bath 5 min

[0244] 6. PBS bath 5 min

[0245] 7. Hematoxylin bath about 20 sec (Harrys Hematoxylin).sup.a).b)

[0246] a) the time depends on the type of hematoxylin used, 20 sec to 10 min (which can be determined by the use of test slides) b) filtrated before use

[0247] 8. Water bath 3 times 3 min

[0248] 9. EtOH 70% bath 5 min

[0249] 10. EtOH 95% bath 5 min

[0250] 11. EtOH 100% bath 5 min

[0251] 12. Xylen bath 5 min

[0252] 13. Xylen bath 5 min

[0253] 14. Mount slide using Permount and coveslip.

[0254] Results

[0255] The effect of treatment with a modified osteopontin polypeptide (SEQ ID NO:1) on hair follicle density is summarised in Table 1(a) and (b) below.

TABLE-US-00017 TABLE 1(a) Polypeptide-treated animals Number of hair Length of follicles epidermis, mm Polypeptide treated Slide 1 area a1 18 0.90 area a2 21 0.90 area a3 25 0.95 area b1 13 0.90 area b2 21 0.90 area b3 26 0.95 Sum 124 5.50 Polypeptide treated Slide 2 area a1 16 0.90 area a2 18 0.90 area a3 25 0.95 area b1 14 0.90 area b2 18 0.90 area b3 19 0.95 Sum 110 5.50 Polypeptide treated Slide 3 area a1 18 0.90 area a2 13 0.90 area a3 19 0.95 area b1 20 0.90 area b2 17 0.90 area b3 14 0.90 Sum 101 5.45 Polypeptide treated Slide 4 area a1 18 0.90 area a2 17 0.90 area a3 15 0.90 area b1 18 0.90 area b2 17 0.90 area b3 13 0.90 Sum 98 5.40 Polypeptide treated Slide5 area a1 13 0.90 area a2 10 0.90 area a3 21 0.90 area b1 12 0.90 area b2 10 0.90 area b3 16 0.90 Sum 82 5.40 Polypeptide treated Slide 6 area a1 18 0.90 area a2 10 0.90 area a3 10 0.90 area b1 18 0.90 area b2 17 0.90 area b3 10 0.90 Sum 83 5.40 Polypeptide treated Slide7 Slide 34 124 5.50 Slide 38 110 5.50 Slide 42 101 5.45 Slide 46 98 5.40 Slide 50 82 5.40 Slide 54 83 5.40 Total 598 32.65

TABLE-US-00018 TABLE 1b Control-treated animals Number of hair follicles Length of epidermis, mm Control treated Slide 1 area a1 9 0.90 area a2 17 0.90 area a3 7 0.90 area b1 11 0.90 area b2 14 0.95 area b3 10 0.90 Sum 68 5.45 Control treated Slide 2 area a1 12 0.90 area a2 17 0.90 area a3 7 0.90 area b1 12 0.90 area b2 13 0.90 area b3 10 0.90 Sum 71 5.40 Control treated Slide 3 area a1 8 0.90 area a2 7 0.90 area a3 11 0.90 area b1 19 0.95 area b2 8 0.90 area b3 14 0.90 Sum 67 5.45 Control treated Slide 4 area a1 21 0.90 area a2 5 0.90 area a3 2 0.90 area b1 16 0.90 area b2 11 0.90 area b3 3 0.90 Sum 58 5.40 Control treated Slide 5 area a1 13 0.90 area a2 2 0.90 area a3 9 0.90 area b1 12 0.90 area b2 7 0.90 area b3 15 0.90 Sum 58 5.40 Control treated Slide 6 area a1 7 0.90 area a2 11 0.90 area a3 10 0.90 area b1 6 0.90 area b2 8 0.90 area b3 15 0.90 Sum 57 5.40 Control treated Slide 7 Slide 18 68 5.45 Slide 22 71 5.40 Slide 26 67 5.45 Slide 34 58 5.40 Slide 38 58 5.40 Slide 42 57 5.40 Total 379 32.50

[0256] A summary of the results is shown below in Table 2

TABLE-US-00019 TABLE 2 No. of follicles No. of follicles analysed per mm Treatment with 356 18.3 SEQ ID NO: 1 Control group 356 11.7

[0257] Representative tissue sections showing follicles in treated and control animals are shown in FIGS. 2 and 3.

[0258] By comparison, wildtype mouse osteopontin was observed to have no detectable effect on hair growth (data not shown).

[0259] Conclusions

[0260] The data show that treatment with the exemplary modified osteopontin polypeptide of SEQ ID NO: 1 increases the number of hair follicles/mm epidermis by about 60%.

[0261] These findings confirm the usefulness of the osteopontin-like polypeptides of the invention in stimulating hair growth.

EXAMPLE B

In Vivo Study of Hair Growth Effect of SEQ ID NOS: 5 and 63 in Mice

[0262] Materials and Methods

[0263] Animals

[0264] Mice (C57BL/6) were used at an age of 6 to 8 weeks.

[0265] Overview of Study Design [0266] Selection of animals in telogen phase of hair growth. [0267] Clipping of dorsal back of animals [0268] Subcutaneous injection of test peptide/vehicle [0269] Visual analysis: Percentage anagen induction; Mean hair growth score; Visual melanogenesis: [0270] Histological analysis: Follicle count in subcutis; Morphometry for skin thickness.

[0271] Treatment Groups [0272] Animals were assigned randomly to treatment groups (see Table 3)

TABLE-US-00020 TABLE 3 No. of Treatment animals Dose Volume Cx 5 60 nM 25 l FOL-004 5 60 nM 25 l (=SEQ ID NO: 5) FOL-005 5 60 nM 25 l (=SEQ ID NO: 63) Vehicle control 5 25 l [0273] Animals were administered the specified treatment on Days 1, 5 and 9 [0274] Treatment were administered by subcutaneous injection into dorsal clipped skin

[0275] Scoring Criteria [0276] The effect of the treatments was observed and scored daily according to the criteria in Table 4

TABLE-US-00021 TABLE 4 Observation of dorsal skin Hair growth score No hair growth, pink skin 0 Skin colour changes from pink to gray without 0.5 visible hair growth Skin colour changes from pink to gray or black 1 without visible hair growth, indicating the onset of anagen Sparse hair growth 1.5 Diffuse short hair growth 2 Moderate hair growth 2.5 Dense, normal coat hair 3

[0277] Results

[0278] The effect of the exemplary polypeptides of the invention on hair growth is shown in Table 5.

TABLE-US-00022 TABLE 5 Day Animal 1 2 5 6 7 8 9 12 13 14 15 16 (A) Vehicle (control) 1 0 0 0 0 0 0 0 0 0 0 NA NA 2 0 0 0 0 0 0 0 0 0 0 NA NA 3 0 0 0 0 0 0 0 0 0 0 NA NA 4 0 0 0 0 0 0 0 0 0 0 NA NA 5 0 0 0 0 0 0 0 0.5 1 1 NA NA Mean 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.2 0.2 (B) Cx (SEQ ID NO: 1 minus the initial 16 amino acid signal peptide) 1 0 0 0 1 1.5 2 2.5 3 3 3 NA NA 2 0 0 0 0.5 0.5 0.5 0.5 1 1 1 NA NA 3 0 0 0 0.5 0.5 0.5 1 1.5 1.5 1.5 NA NA 4 0 0 0 0.5 0.5 0.5 1 1.5 1.5 1.5 NA NA 5 0 0 0 0 0 0 0.5 1 1.5 2 NA NA Mean 0.0 0.0 0.0 0.5 0.6 0.7 1.1 1.6 1.7 1.8 (C) FOL-004 (SEQ ID NO: 5) 1 0 0 0.5 1 1 1 1 1.5 1.5 1.5 2.5 3 2 0 0 2.5 2.5 3 3 3 3 3 3 3 3 3 0 0 2.5 2.5 2.5 3 3 3 3 3 3 3 4 0 0 1.5 2.5 3 3 3 3 3 3 3 3 5 0 0 1.5 2 2.5 2.5 3 3 3 3 3 3 Mean 0.0 0.0 1.7 2.1 2.4 2.5 2.6 2.7 2.7 2.7 2.9 3.0 (D) FOL-005 (SEQ ID NO: 63) 1 0 0 2 2.5 3 3 3 3 3 3 3 3 2 0 0 2 3 3 3 3 3 3 3 3 3 3 0 0 0.5 0.5 1 1 1.5 2.5 2.5 2.5 3 3 4 0 0 2 2.5 2.5 3 3 3 3 3 3 3 5 0 0 0 0 0 0.5 1 2 2.5 2.5 3 3 Mean 0.0 0.0 1.3 1.7 1.9 2.1 2.3 2.7 2.8 2.8 3.0 3.0

[0279] FIG. 4 shows representative photographs of (a) a control-treated animal on day 14 having a hair growth score of 0, (b) an animal treated with exemplary full length polypeptide Cx (SEQ ID NO: 1, 60 nM) on day 14 having a hair growth score of 1.5 (c) an animal treated with exemplary short peptide FOL-004 (SEQ ID NO: 5, 60 nM) on day 5 having a hair growth score of 2.5 and (d) an animal treated with exemplary short peptide FOL-005 (SEQ ID NO: 5, 63 nM) on day 5 having a hair growth score of 2.0.

[0280] The results are summarised in FIG. 5.

[0281] Exemplary polypeptides FOL-004 (SEQ ID NO: 5) and FOL-005 (SEQ ID NO: 63) both induced rapid hair growth, which was evident from day 5 and maintained until the end of the assessment period (day 16).

[0282] A full length polypeptide of the invention, (Cx) also induced pronounced hair growth, albeit with a slower onset than the FOL-004 (SEQ ID NO: 5) or FOL-005 (SEQ ID NO: 63).

[0283] Animals treated with the vehicle control showed little sign of hair growth over the period of the experiment.

[0284] None of the polypeptides tested lead to any adverse effects in the animals (no body weight loss or any other discernible adverse symptom).

[0285] Conclusions

[0286] All mutated osteopontin polypeptides tested showed pronounced hair growth effects in vivo, with the shorter exemplary polypeptides FOL-004 (SEQ ID NO: 5) and FOL-005 (SEQ ID NO: 63) exhibiting a particular fast onset of action.

EXAMPLE C

In Vivo Study of Hair Growth Effect in Mice of the Wildtype Osteopontin Fragment Equivalent to SEQ ID NO: 5

[0287] Materials and Methods

[0288] Animals, as described above in Example B, were treated as described in Table 6:

TABLE-US-00023 TABLE 6 No. of Treatment animals Dose Volume FOL-001* 6 60 nM 25 l (=SEQ ID NO: 121) *FOL-001 consists of the following amino acid sequence:

TABLE-US-00024 [SEQIDNO:121] VDVPNGRGDSLAYGLR [0289] This sequence is a fragment of wildtype mouse osteopontin, and corresponds to the region of the protein from which FOL-004 is derived (i.e. FOL-004 is a mutated version of wildtype fragment FOL-001). [0290] Animals were administered the specified treatment on Days 1, 5 and 9 [0291] Treatment were administered by subcutaneous injection into dorsal clipped skin

[0292] Scoring Criteria [0293] The effect of the treatments was observed and scored daily as described in Example B.

[0294] Results

[0295] The effect of the polypeptide FOL-001 on hair growth is shown in Table 7 and FIG. 6.

TABLE-US-00025 TABLE 7 Day Animal 1 2 5 6 7 8 9 12 13 14 15 16 FOL-001 (SEQ ID NO: 121, 60 nM) 1 0 0 0.5 0.5 0.5 0.5 1 2 2 2.5 2.5 NA 2 0 0 0.5 0.5 0.5 0.5 1 2 2 2.5 2.5 NA 3 0 0 0 0 0 0 0.5 1 1 1 1 NA 4 0 0 0 0 0 0 0 0 0 0 0 NA 5 0 0 0 0 0 0 0.5 1 1 1 1 NA 6 0 0 0 0 0 0 0.5 1 1 1 1 NA Mean 0 0 0.17 0.17 0.17 0.17 0.58 1.17 1.17 1.33 1.33

[0296] Peptide FOL-001 (SEQ ID NO: 121) produced a delayed but detectable hair growth effect in mice, which became evident at about day 9 and reached a plateau at a score of about 1.3.

[0297] Conclusions

[0298] Compared to the corresponding mutated peptide sequence of the invention (FOL-004; SQE ID NO:5), the equivalent non-mutated wild type sequence exhibited a delayed onset of hair growth stimulation with a maximum effect of less than 50% of that observed with FOL-004.

[0299] Thus, the exemplary polypeptide shows much greater efficacy than the equivalent non-mutated wild type sequence.

EXAMPLE D

In Vivo Study of Anagen Induction in Peeled Skin

[0300] Materials and Methods

[0301] Animals and treatments are as described above in Example B.

[0302] All animals were sacrificed on day 16.

[0303] Observation of Melanogenesis

[0304] Following completion of the term of hair growth assessment for Example B, the animals were euthanized and dorsal skin peeled and removed for observation of the internal surface.

[0305] Histological Analysis

[0306] Peeled skin sections were prepared for histological analysis as described in Example A. Follicle count in the subcutis and skin thickness were then measured.

[0307] Results

[0308] Results are summarised in Tables 8 and 9 below.

TABLE-US-00026 TABLE 8 Observation of melanogenesis % Black No. of Skin anagen colour in % anagen animals colour induc- peeled skin induc- showing Group (external) tion* (day 16) tion** hair growth Cx Black (5/5 100 3/5 60 5/5 (60 nM animals) FOL-004 Black (5/5 100 1/5 20 5/5 (60 nM) animals) FOL-005 Black (5/5 100 3/5 60 5/5 (60 nM) animals) Vehicle Pink (4/5 20 1/5 20 1/5 animals) *with respect to external skin colour change from pink to black **with respect to black colour in peeled skin

TABLE-US-00027 TABLE 9 Mean sem No. of animals Follicle count in Group considered subcutis (no.) Skin thickness a (all animals) Cx (60 nM 5 22.20 10.69 308.8 21.94 FOL-004 (60 nM) 5 6.20 5.80 312.2 15.60 FOL-005 (60 nM) 5 34.80 14.58 348.80 40.05 Vehicle 4* 0 0 246.5 8.35 b (animals in anagen only) Cx (60 nM 3/5 36.67 10.69 308.8 21.94 FOL-004 (60 nM) 1/5 29 5.8 312.2 15.60 FOL-005 (60 nM) 3/5 58 14.58 348.80 40.05 Vehicle 0/4* 0 0 246.5 8.35 *Animal No. 5 was found to be a significant outlier (p < 0/05, Grubbs test) and so was ignored in analysis

[0309] The hair growth cycle consists of three phases: a resting telogen phase (C57BL/6 skin is a pale pink colour), an active hair growth anagen phase (where the skin becomes dark gray or black), and finally a catagen phase (where hair growth stops, and the skin transitions back to the telogen phase, returning to a pale pink colour).

[0310] In the present study, the animals were preselected for pink skin (telogen phase) and the test polypeptide was administered by subcutaneous/topical route. A good hair growth promoter triggers the transition from the resting telogen phase to the active anagen phase, and thus a transition from light skin to dark skin. This dark pigmentation may result from the collection of melanin in the hair follicles, in preparation for new hair growth during the anagen phase. Melanin synthesis of follicular melanocytes is strictly coupled to the anagen phase, ceases during catagen and is absent in telogen phase. Hence a good hair growth promoter causes blackening of dorsal skin. Upon termination, the dorsal skin of the animals was then excised out by peeling and the peeled skin reversed and observed for the induction of melanogenesis indicated by visual blackening. Histological analysis reveals number of follicles and skin thickness. A good hair growth promoter increases no. of follicles in sub cutis and skin thickness.

[0311] In the present study, all three test polypeptides led to the appearance of dense hair growth after three subcutaneous injections (see Example B above). It was also observed that in some of the animals, subsequent to full growth of the hair, the skin colour changed from black to gray to pink, a characteristic of catagen phase. The peeled skin collected in the study showed the black patch, indicative of anagen phase (active phase) of hair growth cycle due to melanocyte sequestering. In these animals, the histological results showed a large number of hair follicles and increase in skin thickness. But in some animals, where the catagen phase had occurred, peeled skin was found to be of pink colour, even though the hair coat was intact. Thus, analysis of such peeled skin sections demonstrated lack of follicles in subcutis but still good increase in skin thickness.

[0312] Conclusions

[0313] The exemplary mutated osteopontin polypeptides of the invention showed pronounced induction of anagen, as evidenced by an increase in the number of hair follicles in the subcutis and/or enhanced skin thickness.