Effect of Propecia on the Hair Follicle in Male Androgenetic Alopecia: A Confocal Laser Scanning Microscopy and Video Imaging Study

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1 Effect of Propecia on the Hair Follicle in Male Androgenetic Alopecia: A Confocal Laser Scanning Microscopy and Video Imaging Study

2 Investigators: Department of Dermatology, University of Minnesota, Minneapolis, Minnesota, USA Maria Hordinsky, MD Marna Ericson, PhD Cathy Boeck, RN, DNR, CCRC Dawn Snow, LPN, CCRC Jill Plumb-Smith, BA Christine Baker, BS Medical School Grants Program, Merck & Co., Inc.

3 Objective Characterize changes in hair follicle innervation, vasculature,, and melanocytes before and after administration of Propecia (1 mg/day) or placebo for 9 months.

4 Study Design Patients Treatment Efficacy Analysis of nerves, vessels, and melanocytes 15 Caucasians and 1 African-American American with Hamilton type IV or V balding, between the ages years Propecia 1 mg po qd or placebo, 1 tablet po qd, for nine months Clinical photographs, investigator assessment, analysis of 4-mm 4 scalp punch biopsies by H & E and LSCM Immunohistochemistry and laser scanning confocal microscopy (LSCM)

5 Study Design Patients Treatment Efficacy Analysis of nerves, vessels, and melanocytes 15 Caucasians and 1 African-American American with Hamilton type IV or V balding, between the ages years Propecia 1 mg po qd or placebo, 1 tablet po qd, for nine months Clinical photographs, investigator assessment, analysis of 4-mm 4 scalp punch biopsies by H & E and LSCM Immunohistochemistry and laser scanning confocal microscopy (LSCM)

6 Study Design Patients Treatment Efficacy Analysis of nerves, vessels, and melanocytes 15 Caucasians and 1 African-American American with Hamilton type IV or V balding, between the ages years Propecia 1 mg po qd or placebo, 1 tablet po qd, for nine months Clinical photographs, investigator assessment, analysis of 4-mm 4 scalp punch biopsies by H & E and LSCM Immunohistochemistry and laser scanning confocal microscopy (LSCM)

7 Study Design Patients Treatment Efficacy Analysis of nerves, vessels, and melanocytes 15 Caucasians and 1 African-American American with Hamilton type IV or V balding, between the ages years Propecia 1 mg po qd or placebo, 1 tablet po qd, for nine months Clinical photographs, investigator assessment, analysis of 4-mm 4 scalp punch biopsies by H & E and LSCM Immunohistochemistry and laser scanning confocal microscopy (LSCM)

8 Pre- and Post-Treatment Analyses 1. Clinical photographs 1. Vertex 2. Anterior 2. Histology 3. Investigator assessment 4. LSCM merged z-series z images of multi-stained biopsy sections

9 Pre-Treatment Post-Treatment Hamilton Type IV Hamilton Type IV Placebo +/06B

10 Pre-Treatment Post-Treatment Placebo +/06B

11 Pre-Treatment Post-Treatment Hamilton Type IV Hamilton Type III Propecia +/01A

12 Pre-Treatment Post-Treatment Propecia +/01A

13 Pre- and Post-Treatment Analyses 1. Clinical photographs 1. Vertex 2. Anterior 2. Histology 3. Investigator assessment 4. LSCM merged z-series z images of multi-stained biopsy sections

14 Pre-Treatment Post-Treatment Placebo +/06B

15 Pre-Treatment Post-Treatment OBSERVATION: Follicles deeper in dermis/fat Propecia +/01A

16 Pre- and Post-Treatment Analyses 1. Clinical photographs 1. Vertex 2. Anterior 2. Histology 3. Investigator assessment 4. LSCM merged z-series z images of multi-stained biopsy sections vertically-sectioned microns thick

17 Immunohistochemical Epifluorescent Techniques Nerve marker Polyclonal antibody to pan- neuronal cytoplasmic protein gene product 9.5 (PGP 9.5) and anti-rabbit Cy 5 or Cy 3 Melanocytes Melanocytes Monoclonal antibody Mel 5 and anti-mouse Cy 3 or Cy 5 Vasculature and follicular keratinocytes Fluorescein-conjugated conjugated lectin Ulex europaeus agglutinin (UEA1-FITC)

18 Laser Scanning Confocal Microscopy (LSCM) LSCM differs d from conventional wide-field microscopy because: Out-of of-focus focus epifluorescent light is excluded,, thereby increasing the contrast and resolution in the plane of focus. Images from defined levels, in the z-plane z of focus, can be captured and stored in the computer's memory and projected in register. Thick sections (up to 400 microns) can be imaged so that the entire, intact hair follicle is captured.

19 Laser Scanning Confocal Microscopy (LSCM) Captures 3-Dimensional Data of Intact Cellular Structures and Interactions Bottom optical section in z-plane of sample. Top optical section in z-plane of sample. Confocal microscope records digital image of each optical section. Digitized layers merged into single in-focus projection.

20 examples

21 Normal Scalp Anagen Hair Follicle

22 Normal Scalp Anagen Hair Follicle Montage of four LSCM z-series fields

23 Montage of four LSCM z-series fields Normal Scalp Anagen Hair Follicle PGP 9.5 antibody labels nerves

24 Montage of four LSCM z-series fields Normal Scalp Anagen Hair Follicle PGP 9.5 antibody labels nerves UEA1-FITC stains: blood vessels follicular keratinocytes subset of epidermal keratinocytes

25

26 Bulge region of normal anagen scalp hair follicle.

27 Bulge region of normal anagen scalp hair follicle. PGP9.5/Nerves

28

29 UEA 1/Vessels Bulge region of normal anagen scalp hair follicle

30 Bulge region of normal anagen scalp hair follicle. CGRP

31 UEA 1/Vessels PGP9.5/Nerves CGRP

32 UEA 1/Vessels Bulge region of normal anagen scalp hair follicle. PGP9.5/Nerves

33 Bulge region of normal anagen scalp hair follicle. PGP9.5/Nerves CGRP/Neuropeptide

34

35 UEA1-FITC Staining of Perivascular Network Surrounding Bulb Region of Normal Scalp Anagen Hair Follicle

36

37 UEA1-FITC Staining of Vasculature and Follicular Keratinocytes in and around Dermal Papilla of Normal Scalp Anagen Hair Follicle

38 Pre- and Post-Treatment Analyses 1. Clinical photographs Vertex Anterior 2. Histology 3. Investigator assessment 4. LSCM merged z-series z images Melanocytes blue color Vessels and follicles red color Nerves green color

39

40 Pre- Treatment

41 Pre- Treatment Merged LSCM z-series of Multi- Stained Scalp Biopsy

42 Pre- Treatment Miniaturized Follicles Merged LSCM z-series of Multi- Stained Scalp Biopsy

43 Pre- Treatment area of higher magnification Miniaturized Follicles Merged LSCM z-series of Multi- Stained Scalp Biopsy

44 Merged LSCM z-series of Multi- Stained Scalp Biopsy Pre- Treatment

45 Pre- Treatment

46 Pre- Treatment UEA1-FITC stains: blood vessels follicular keratinocytes subset of epidermal keratinocytes

47 Pre- Treatment

48 Pre- Treatment PGP 9.5 antibody labels nerves.

49 Pre- Treatment

50 Pre- Treatment Mel 5 antibody labels epidermal and follicular melanocytes.

51 Pre- Treatment 3-D D Animation of Merged LSCM z-seriesz

52 Post- Treatment

53 Post- Treatment Merged LSCM z-series of Multi- Stained Scalp Biopsy

54 Post- Treatment area of higher magnification

55

56 Post- Treatment Innervation of Putative Stem Cell/Bulge Region.

57

58 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal

59 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal

60 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal

61 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal

62 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal

63 Summary Propecia induced hair regrowth. After nine months Propecia therapy, LSCM images of multi-stained biopsies revealed: enlarged follicle diameters follicles deeper in dermis/fat perifollicular innervation comparable to normal Mel5 antibody staining in the dermal papilla comparable to normal..qualitative

64 Continued Analysis Quantitative analysis of melanocytes in hair bulb all subjects, before and after treatment, using stereological analysis of three-dimensional LSCM z-series images.

65

66

67 Transplant Proposal Demonstrate that nerves and blood vessels in transplanted hair follicles become integrated into the dermal nerve and vasculature plexus of the recipient site, respectively, after 3 months post surgery. Demonstrate increased VEGF, FKBP12, SP, and NGF in and around the transplanted hair follicles at 2 weeks post surgery versus pre-surgery and 3 months post-surgery.

68 Transplant Proposal Demonstrate that nerves and blood vessels in transplanted hair follicles become integrated into the dermal nerve and vasculature plexus of the recipient site, respectively, after 3 months post surgery. Structure Demonstrate increased VEGF, FKBP12, SP, and NGF in and around the transplanted hair follicles at 2 weeks post surgery versus pre-surgery and 3 months post-surgery.

69 Transplant Proposal Demonstrate that nerves and blood vessels in transplanted hair follicles become integrated into the dermal nerve and vasculature plexus of the recipient site, respectively, after 3 months post surgery. Structure Demonstrate increased VEGF, FKBP12, SP, and NGF in and around the transplanted hair follicles at 2 weeks post surgery versus pre-surgery and 3 months post-surgery.

70 Transplant Proposal Demonstrate that nerves and blood vessels in transplanted hair follicles become integrated into the dermal nerve and vasculature plexus of the recipient site, respectively, after 3 months post surgery. Structure Demonstrate increased VEGF, FKBP12, SP, and NGF in and around the transplanted hair follicles at 2 weeks post surgery versus pre-surgery and 3 months post-surgery. Growth Factors and Endogenous Neurotrophic Compounds

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