Ive read through some of the patent, and paid attention to the experimental details section. It seems to me that they are dermabrating the skin with a felt wheel (both human skin grafted onto mice, and mouse skin), and SUPPRESSING WNT EXPRESSION FOR NINE DAYS EXACTLY, and then just watching the new PIGMENTEDfollicles grow in.
If wnt epression is not blocked (they used DKK-1, which inhibits wnt) for the first nine days, the hairs will lack pigment during this “epilithealization” period.
Maybe Im reading it wrong, but I dont’ see where wnt is ever specifically applied or a analogue or wnt-mimicing subtance thereof. Apparently the wounding gets the body to make it enough on its own. If someone reads the patent and sees where something is being used to induce wnt-pathways after the first nine days, please let me know.
The expiremental details are below:
EXPERIMENTAL DETAILS SECTION
EXAMPLE 1
DEPILATION AND EPIDERMAL ABRASION CAUSES DE NOVO HAIR FOLLICLE
FORMATION
MATERIALS AND EXPERIMENTAL METHODS
Depilation and epidermal abrasion
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[000187] Mice were anesthetized with an injection of sodium pentobarbital before the hair on the back was clipped and depilated withNair (Carter-Wallace, New York, NY), then epidermis was removed using a rotating felt wheel as described by Argyris T, J Invest Dermatol, 75: 360-362, 1980). After scrubbing with 70% ethanol and drying under an incandescent lamp, the basal and supra-basal layers in an area of (1.5 cm)2 cm of the inter-folHcular epidermis were removed by careful abrasion with a felt wheel mounted on a Dremel Moto-tool (Racine, WI). After abrasion, the skin was shiny and smooth, and there was no blood. One day later, the abraded area was covered by a fibrin crust, which fell off after 3-7 days, exposing the newly regenerated epidermis. A group of control mice was sacrificed immediately after abrasion to confirm microscopically the complete removal of the interfollicular epidermis.
Immunohistochemistry
[000188] Skin samples were fixed in PBS-buffered 10% formalin. Six-micron thick paraffin sections were cut and stained, where applicable, with antibodies.
BrdU labeling [000189] The protocol described by Bickenbach and colleagues (Bickenbach et al, Cell Tiss Kinet 19: 325-333, 1986; Bickenbach et al, Exp Cell Res 244. 184-195, 1998) was used. Mice were injected with 50 milligrams per kilogram (mg/kg) bodyweight 5-bromo-2’-deoxyuridine (BrdU) every 12 hours for a total of four injections.
RESULTS
[000190] An area of the backs of 50-day old mice was subjected to depilation and removal of the epidermis using a rotating felt wheel. Fifteen days later, HF placodes, hair germs and other signs of follicle neogenesis were present (Figure 1 ; arrow indicates a hair germ). Morphology of the follicles was similar to embryonic follicle development. To further characterize proliferation in the new follicles, the skin was labeled with BrdU 60 minutes before sacrifice. As depicted in Figure 2, the proliferation pattern was similar to developing follicles in the embryo.
[000191] These findings demonstrate that (a) disruption of the epidermis causes generation of new
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HF, and that this generation of new HF can occur (b) in adult subjects and (c) during telogen (50- day-old mice are in the second telogen stage of the hair cycle).
EXAMPLE 2
INDUCTION OF A LARGE EXCISIONAL WOUND, BUT NOT A SMALL PUNCH WOUND, CAUSES DE NOVO HAIR FOLLICLE FORMATION
MATERIALS AND EXPERIMENTAL METHODS
Punch wound and excisional wound induction
[000192] The backs of 21 -day-old mice were depilated as described for Example 1 and sterilized with alcohol, followed by 1% iodine solution. Punch wounds, 4 mm in diameter, were induced using a dermal biopsy punch, down to, but not through, the muscle fascia. Excisional wounds were full thickness and 1 cm in diameter; skin and panniculus carnosus was excised using fine surgical scissors.
RESULTS
[000193] To test whether wounding could induce HF formation, punch wounds or excisional wounds were induced in mice. Both types of wounds exhibited contraction and re-epithelialization following wound induction; however, unlike the mice receiving punch wounds, the mice receiving excisional wounds also exhibited scar formation within 10 days of wound induction (Figure 3, left panel). No follicles were evident at this time point (Figure 3, right panel). 12 days after wound induction, hair germs, with similar morphology to fetal hair germs, were observed in the wound site, following BrdU pulse labeling (Figure 4). Several markers were used to verify that the observed structures were HF. The structures exhibited staining with anti-keratin 17 (K 17), an HF marker (Figure 5), and staining with anti-alkaline phosphatase at the 12 day time point verified that the structures had dermal papilli containing fibroblasts, as expected for HF (Figure 6; HF at earlier and later stages are depicted in the left and right panels, respectively).
[000194] The HF generated by wound induction were further characterized by morphological
P-7628-PC comparison to embryonic HF9 following BrdU staining; a clear correspondence in morphology was observed at various stages (Figure 7). In addition., several markers of embryonic HF development, namely Left, wingless/ int (Wnt) 10b, and sonic hedgehog (Shh), were also induced in the epidermal disruption-induced HF neogenesis (EDIHN) (Figure 8). Additional BrdU staining (Figure 9) and staining for HF markers S 100A3 and S 100A6 (Figure 10; left panel: tissue section parallel to HF axis; right panel: cross-sectional view of follicle) provided further verification that the development of the EDIHN follicles closely paralleled embryonic HF development.
[000195] These findings provide further evidence that disruption of the epidermis causes generation of new HF, and that this generation of new HF can occur (b) in adult subjects and (c) during telogen (21 -day-old mice are in the first telogen stage of the hair cycle).
EXAMPLE 3
EDIHN-INDUCED HAIR FOLLICLES GENERATE HAIRS
[000196] At 25 and 45 days after wound induction, wound sites contained new hairs (Figure 11 „ left and right panels, respectively). New hairs appeared to lack pigmentation, except when the wnt pathway was inhibited, using Dkk-1 (Dickkopf-1) during the first nine days after wounding (see Example 10).
[000197] These findings indicate that EDIHN-induced HF function normally; Le. are capable of generating hairs.
EXAMPLE 4
EDIHN HAIR FOLLICLES RETAIN THE ABILITY TO ENTER INTO CYCLICAL
HAHl GROWTH
MATERIALS AND EXPERIMENTAL METHODS
BrdU labeling
[000198] 50 mg/kg bodyweight BrdU (Sigma) was injected twice per day for 3 days beginning 20
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Whole mounting and immunofluorescence
[000199] HF whole mounts were obtained by incubating fresh skin with EDTA (2OmM in PBS) at 37
0C overnight, then separating the epidermis and dermis. Epidermis was then fixed in 10% formalin for 10 min, room temperature (RT). Dermis was fixed in acetone overnight, RT. After rinsing with
PBS, whole mounts were stained with antibodies for immunohistochemistry (schematically depicted in Figure 12) and were imaged using a Leica confocal microscope.
RESULTS
[000200] To determine whether EDIHN-induced HF contain normal levels of HF stem cells, mouse skin was examined for the presence of label-retaining cells at 21 days after wound induction.
Retention of BrdU during a long chase period is, under these conditions, one of the hallmarks of HF stem cells. Normal numbers and placement of label-retaining cells (in the bulge of the HF) were observed (Figure 13). To verify that the label-retaining cells were HF stem cells, K15-eGFP mice were utilized. In these mice, eGFP (enhanced green fluorescent protein) is expressed from the Kl 5 promoter; thus, expression of eGFP identifies HF stem cells. As depicted in Figure 14A5 eGFP- expressing cells were observed in in tissue sections (right side) of newly formed hair follicles 35 days following wound induction. eGFP-expressing cells were also seen in the epidermis whole mounts (bottom, far left panel) indicating the conversion of epidermal cells into cells with hair follicle stem cell characteristics, ([bottom, second from left] panel is same as [bottom, far left] panel but viewed under white light) This finding shows that the observed label-retaining cells exhibited
HF stem cell properties.
[00020I] To determine whether EDIHN-induced HF cycle normally, mounts were prepared from additional mice at 35, 38 and 45 days after wounding. As depicted in Figure 14B, the EDIHN- induced HF entered the resting phase, telogen, and then re-entered a new anagen stage.
[000202] In summary, the findings of this Example show that EDIHN-induced HF contain HF stem cells, as do embryonically generated HF. The presence of the HF stem cells shows that EDIHN-
P-7628-PC induced HF retain the ability to enter into cyclical hair growth in the same manner as embryonically generated HF. The findings also show that wounding induces epidermal cells to assume a hair follicle stem cell state (expressing Kl 5-eGFP). This model is shown schematically in Figure 15. The findings of Examples 2, 3, and 4 showthat EDIHN-induced HF are fully functional and thus able to restore hair growth to a subject in need.
EXAMPLE 5
EPIHN-INDUCES NEW HAIR FOLLICLES IN MICE AT THE TELOGEN STAGE OF
THE HAIR CYCLE
[000203] To determine whether EDIHN was induced new hair follicles in mice wounded at the telogen stage of the hair cycle, 21 -day-old mice were subj ected to ED3ΗN using a 1 -cm excisional wound, as described in Example 2, Sldn was then examined by whole-mount assay for indications of new HF. As depicted in Figure 16, after 11 days, new HF were not evident by macroscopic examination (top panel), AP staining of the dermis (bottom left panel), or KI 7 staining of the epidermis (bottom right panel). After 14 days, as depicted in Figure 17, dermal papilla cells were detected in the dermis (left panel) and HF stem cells in the epidermis (right panel), demonstrating that new follicles were being formed. After 17 days, the new follicles were more developed, as shown by examination of the dermis and epidermis (Figure 18, left and right panels, respectively). This method induced formation of an average of 49 new follicles in the wound, a number that was consistent over three separate experiments, as depicted in Table 1.
[000204] Table 1. Results of three separate experiments performed on 21 -day-old mice.
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[000205] The findings of this Example demonstrate that EDIHN is capable of inducing formation of new HF in mice at the telogen stage of the hair cycle, despite that fact that these mice do not contain HF at the anagen stage during wounding.
EXAMPLE 6
IN ADULT MICE. INDUCTION OF ANAGEN INCREASES THE EFFICIENCY OF
EDIHN
[000206] The experiment described in Example 5 was repeated with mice of different ages, and therefore at different stages of the hair cycle. To ensure that wound scarring occurred, larger wounds were in induced in the older mice. As depicted in Table 2, adult mice at telogen, such as 8-week-old mice, exhibited lower efficiencies of HF formation by EDIHN.
[000207] Table 2. Efficiency of HF formation by EDIHN in adult mice at various stages of the hair cycle.
- The second telogen lasts approximately 40 days in mice. Thus, 14-week-old and 20- week-old mice contained a mixture of telogen and anagen HF.
[000208] To determine whether experimental induction of anagen increased the efficiency of EDIHN,
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8-week-old mice were depilated several days prior to wound induction. As depicted in Figure 19, the wounds closed similarly whether or not they were preceded by depilation. As depicted in Figure 20A-B, the depilated mice exhibited enhanced EDIHN relative to the non-depilated mice depicted in the previous Example by a factor of 11 -fold.
[000209] The findings of this Example demonstrate that anagen induction enhances EDIHN. In addition, these findings show that EDIHN is capable of not only forming new HF, but also of activating anagen in pre-existing HF in the telogen stage.
EXAMPLE 7
EDIHN-INDUCES NEW HAIR FOLLICLES IN HUMAN SKIN
MATERIALS AND EXPERIMENTAL METHODS
Grafting
[000210] Discarded human, adult scalp from the preauricular area obtained from plastic surgery was grafted onto immunodeficient (scid) mice. The graft was bandaged and allowed to heal, then was used in the wound healing study 3 months after grafting.
RESULTS
[000211 ] To determine whether human skin responded to EDIHN as did mouse skin, human skin was grafted onto SCID (immuno-deficient) mice and subjected to depilation by plucking and wound induction three days later. Seven days following wound induction, formation of new HF was observed in the human skin (Figure 2 IA; arrows indicate new HF) by hematoxylin and eosin staining of paraffin embedded tissue sections.
[000212] In additional experiments, adult human skin was grafted onto mice., abraded, and examined at 7 days post-abrasion. New HF were generated in the human skkx, which mimicked normal hair follicle formation during fetal development, as evidenced by staining for SlOO A6 or S100A4 (Figure 21B).
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[000213] The results of this Example show that EDIHN can be used to generate hair growth inhuman skin as for mouse skin.
EXAMPLE 8
MOLECULAR PATHWAYS ACTIVATED DURING HF STEM CELL ACTIVATION
MATERIALS AND EXPERIMENTAL METHODS
Isolation and activation of HF stem cells
[000214] Kl 5~eGFP mice were depliated in order to induce formation of new HF. Activated hair follicle stem cells were isolated from K15-eGFP mice using fluorescence-activated cell sorting (FACS) two days after depilation and 5 μg (micrograms) total RNA from the cell population was isolated, reverse-transcribed and hybridized to an Affymetπx (Santa Clara, California) array MG_U74v2 chip. Scanned chip images were analyzed using Affymetrix Microarray Suite 5.0 and GeneSpring software (Silicon Genetics) to detect fold-change differences between activated HF stem cells (HFSCs) and non-activated (telogen) HFSCs. Values were normalized before computing fold-changes and differences between non-activated “bs-line” and activated (“expt”) samples.
RESULTS
[000215] To identify molecular pathways up-regulated during HF stem cell activation, activated HF stem cells were isolated, and the gene expression patterns of the cells were analyzed to detect up- regulated transcripts. The transcripts depicted in Table 3 were up-regulated at least 2~fold in the activated HF stem cells relative to the cells prior to activation. In some cases, the sequence in Table 3 is a genomic sequence that contains the sequence of the transcript. Data pertaining to the up- regulation of the transcripts and further information about them is provided in Figure 22.
Me again…
here is the patent page, WIPO - Search International and National Patent Collections ,
I dont see where anything external (other than delipidation done with conventional NAIR, which was admittedly not really necessary for effect) was done other than the blockage of wnt for nine days. The description would seem to indicate that they are prepared for ORAL administration of the wnt-‘blocker’ by the verbiage below
000181] The pharmaceutical compositions containing the HF stem cell-inducing or -activating compound can, in another embodiment, be administered to a subject by any method known to a person skilled in the art, such as topically, parenterally, paracancerally, transmucosally, transdermally. intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially, intravaginally or intratumorally. Each possibility represents a separate embodiment of the present invention.
[000182] In another embodiment, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In one embodiment of the present invention;, the HF stem cell-inducing or -activating compounds are formulated in a capsule, hi another embodiment, the compositions of the present invention comprise, in addition to the HF stem cell-inducing or -activating compound active compound and the inert carrier or diluent, a hard gelating capsule.
Green tea catechin ECGC and curcumoids both inhibit wnt expression. I keep looking into the patent to see where something that activates wnt comes in, but haven’t noted it yet. If anyone does, let me know. It appears at this point they are merely dermabrating skin over five millimeters in width and just deep enough to remove its top layer (but the experimtal subjects did NOT bleed), and blocking wnt for 9 days, and voila’…new DARK hair.
Surely it can’t be that easy.
I’d like to note that they had the same success in human skin on mousebacks as they did with mere mouseskin.
but also in human skin grafts on mice.