Ziering C, Zimber MP, Zeigler F, Hubka M, Mansbridge JP, Hubka K, Perez-Meza D, Naughton GK
Introduction
Androgenetic alopecia is a widespread cosmetic and medical disorder for which there exist few treatment options. The current therapeutic strategies including surgical, pharmaceutical and cosmetic interventions are limited in approach and success. We have developed a bioengineered human cell-derived formulation, termed Hair Stimulating Complex (HSC), that consists of a number of human growth factors and morphogens recognized to be critical to the induction and maintenance of hair follicle growth and activity. Here we report that the preparation is safe as applied and showed effectiveness in stimulating hair growth in the C57/Bl6 mouse model as well as in an exploratory first-in-human, Phase I clinical study in men with male pattern baldness.
In the HSC manufacturing process, neonatal dermal fibroblasts , which are closely related to hair follicle dermal papilla cells, are grown under hypoxic conditions of 3-5% oxygen, which is characteristic of the embryonic environment. Under these conditions the cells differentially express over 5000 genes compared to cells grown in normoxic environments. Several of the upregulated genes expressed in the hypoxic cells are associated with pluripotent and follicular stem cells including LnX2, SOX21, Nestin, NFATc1, Krt15, POU5F1 (OCT4), SOX2 and Nanog. In addition, stem cell markers are evident on the cells grown in the hypoxic bioreactors, including Nodal, brachyury, Nestin, and Oct 4 (Figure 1).
Figure 1: Hypoxic neonatal cells stained for stem cell-associated proteins (green) and counterstained with phalloidin (red) and DAPI (blue) to demonstrate the cytoskeleton (actin) and nuclei, respectively.
A. Nodal; B. Brachyury; C. Nestin; D. Oct4 (HRP detection system). Mag =40x
These multipotent cells also secrete factors that have been shown to be key in stimulating hair growth, including Wnt 7a, VEGF, KGF and follistatin. In the adult, Wnt proteins have been found to play an essential role in induction of the dermal papilla ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":13903}]} 1, ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":8981}]} 2 and the triggering of stem cell activity in keratinocytes ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":4771}]} 3 to produce new hair follicles and growth. Follistatin is an antagonist of activin and BMP, which are involved in maintaining a slow cycling stem cell phenotype in resting hair follicles ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":174}]} 4. KGF and VEGF have also been shown to be important in normal hair growth and follicle maintenance. The exogenous administration of Wnt proteins and follistatin to the scalp represents a novel and practical way to ameliorate and reverse androgenetic alopecia and other related hair loss disorders. The presence of Wnt proteins in the HSC used in these studies was assessed by immunoblot analysis using a primary antibody recognizing the Wnt7a protein (Santa Cruz Biologicals, CA). The canonical Wnt bioactivity of HSC was confirmed by demonstrating nuclear translocation of -catenin in human epidermal keratinocytes in vitro. LiCl was used as a positive control ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":7829}]} 7, and the Wnt receptor antagonist DKK-1 prevented -catenin translocation.
Preclinical studies demonstrated no safety issues and suggested that the induction of anagen in telogen follicles in a murine model of hair growth might be accelerated by injection of HSC ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":14515}]} 5, ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":6675}]} 6. Over 80 C57Bl/6 mice were injected with either HSC or non-conditioned media and followed for 12 weeks to topically assess new hair growth and pigmentation and the total number of hair follicles at the injection site. An average of 75% of the HSC treated mice showed new hair growth at the injection site (Figure 2) as well as a 50% increase in the number of new hair follicles whereas only 20% of the control mice showed hair growth, most likely due to wounding during the shaving process.
Figure 2: HSC was evaluated in a murine model based on the cyclical pattern of hair growth observed in the C57BL/6 mouse. Animals that were shaved and confirmed to be in a telogen phase of hair growth were given a single intradermal injection of HSC was administered. A. Control animals that received vehicle alone did not demonstrate significant hair growth (white arrow = mechanically-induced hair growth). B. HSC-treated animals showed hair growth in 75% of the animals.
Preclinical safety studies have been performed to evaluate the potential toxicity resulting from administration of HSC using established experimental models in rats. Selection of the rat as an acceptable relevant species is due to the efficacy of the test article observed in rodent models, and the biological homology of the targeted mechanisms of actions shared between mammalian species, including human. The studies were performed by third-party contract research organizations in a manner consistent with standards of GLPs and applicable regulatory and animal welfare compliance. An initial, single-dose acute toxicity study was performed in which animals receiving a single, intradermal injection of HSC were assessed for toxicity by clinical observations of the injection sites (Draize assessment) and general morbidity during the in-life phase, followed by hematology, blood clinical chemistry and post-mortem macroscopic and microscopic pathology at the end of the 8 day study.
The results of this study indicate that HSC did not produce significant toxicity when administered as a single subcutaneous injection to Sprague Dawley rats. To allow multiple administrations of HSC in the clinical setting, a second preclinical study was performed that evaluated toxicity of repeated-dose administration of HSC in rats. Animals received multiple doses of HSC or control at weekly intervals for a duration of 6 weeks. Animals were euthanized either 24 hours or at 4 weeks following the final administration of test article. Toxicity was determined using similar clinical, blood analysis and pathological observations as was performed in the single-dose study and also included ophthalmic exam and urinalysis. In summary, no adverse events were noted in any treatment groups during either of the studies and the results of the subsequent clinical and pathological analysis concluded that the HSC formulations did not produce significant toxicity in rats when administered in a single-dose or a repeated-dose paradigm.
Methods
A pilot clinical trial was undertaken to assess the safety and efficacy of HSC in man. The study design was a single site, double-blind, randomized, placebo-controlled, clinical trial. Two HSC preparations were evaluated in the study; a 10x concentrated, serum-free preparation, and a second, non-concentrated, bovine serum-containing preparation. Also as part of the study, we evaluated whether additional stimulation to the scalp prior to HSC injection would have any effect. Three different devices were used to stimulate the scalp: 1) micro-dermabrasion (MegaPeel, DermaMed Intl, Inc. , Lenni, PA), 2) overlapping passes of nonablative 1540 and ablative 2940 erbium laser (Palomar Medical Technologies, Inc. , Burlington, MA), 3) low level light therapy by the Revage670 (Apira Science Inc. , Newport Beach, CA).
After obtaining informed consent, 26 healthy male subjects between 18-55 yrs of age were enrolled. Inclusion criteria included a Fitzpatrick score of I-IV, Norwood/Hamilton Classification for male pattern hair loss 4-6, and no history of prior hair treatments or immunological compromise. The selected study participants were randomly assigned to one of the three stimulation methods. Four zones, two anterior and two posterior, were selected as treatment sites on each subject’s scalp and marked by a tattoo on its periphery to identify location. The anterior two sites were randomized right/left and injected with one of the two HSC preparations or placebo (unconditioned medium) with no pre-injection stimulation (Fig. 3). The posterior treatment sites were stimulated using one of the three treatments mentioned above followed immediately by injection with HSC or placebo, also randomized right/left. In all subjects, baseline measurements were obtained and each site received four evenly placed intradermal 0.1 cc injections. The ability to randomize and provide different, independent treatment at each of the 4 sites on each subject is a unique and advantageous clinical paradigm in evaluating hair restoration strategies. In addition to comparing treatment and placebo effects throughout the study in individual subjects, thereby providing in-subject control, pretreatment baseline measurements provides greater statistical power by enabling a repeated-measures experimental design.
Figure 3: Double-blind, placebo controlled, randomized, each subject acts as their own control.
In 6 patients, punch biopsies (4 mm) were performed at baseline and week 22 and were used for histopathological evaluation. Biopsies were taken from 6 further patients at an optional 1 year visit. Safety outcomes were also measured through visual examination by the clinician at each time point for inflammation, redness, edema, itching, burning, swelling and any other adverse events. Global and macro photography was reviewed by independent dermatologists for any observable adverse events including redness, swelling and ingrown hairs. The biological and clinical effects of treatment were evaluated through FotoFinder Xpert Clinical Trial System (FotoFinder Systems, Columbia, MD) image acquisition and TrichoScan analysis for anagen:telogen ratios, hair diameter, hair density, hair thickness, and vellus and nonvellus, terminal hair counts. Results are expressed as group means + SEM. The equivalence of sites at baseline, prior to treatment, was determined using one-way ANOVA. Subsequent differences between group means within a treatment group at 12, 22, and 52 weeks post-treatment were evaluated using paired (repeated-measures), two-tailed Student’s t-tests. All other differences between group means were evaluated using unpaired, two-tailed Student’s t-tests. Statistical significance was set at level of = 0.05. A comparison of the untreated and treated baseline mean hair measurements detected no statistically significant differences, so any subsequent differences could be inferred to be the result of experimental treatment. A combination of investigator global assessments and test subject self-assessments were also performed at the times of clinical visits. Investigators used a rating system to determine visual improvement in regional hair growth.
Results
The primary objective of the study was to assess clinical safety of HSC administration in human subjects. All patients tolerated the procedures well and no significant complaints, clinical symptoms or signs of an adverse reaction were reported in any subjects. The histopathological evaluation of the treatment site biopsies taken at baseline, 22 weeks and 1 year post-treatment revealed minimal to slight inflammation at the injection site, and no abnormal morphology, hamartomas or other pathological responses. From these observations, it is concluded that a single, intradermal injection of HSC did not result in any significant toxicity, pathology or other adverse event during the course of the study.
For the secondary objective, to assess the clinical effect of HSC, the sites treated with 10X, serum-free HSC (n=13) demonstrated an increase in most hair growth indicators over the initial 12 week evaluation period. A statistically significant increase from baseline values in the hair shaft thickness (p = 0.04), hair thickness density ( p = 0.025) and the number of non-vellus, terminal hairs (p = 0.003) was observed in the HSC treated sites (Fig. 1E). The improvements caused by HSC treatment were significantly greater than those observed in placebo treated sites for hair shaft thickness (6.3% + 2.5% vs. -0.63% + 2.1%; p = 0.046), thickness density (12.8% + 4.5% vs. -0.2% + 2.9%; p = 0.028), and terminal hair density (20.6 + 4.9% vs. 4.4 + 4.9%; p = 0.029). Although a similar trend was seen at 22 weeks, significance was lost as there was no further growth improvement in the subjects. However, at 1 year, we observed significant improvements in hair count (16.0 +6.6% vs. 3.65 + 3.7%, p=0.032) and substantial increases in thickness density (17.6 +8.39% versus 0.67 + 4.3%), and terminal hair density (29.5 + 14.8 versus 2.4 + 6.8%).
Figure 4: Hair growth improvement observed in one subject 12 months after single administration of HSC. Hair count increase from 179 at baseline to 263.5 at 12 months, with a 73.61% increase in terminal hairs, was observed.
Placebo treated sites (n=12) at 12, 22 and 52 weeks showed no significant improvements in any of the measured hair growth indicators. In addition, no significant effects were seen with the serum containing HSC preparation. The additional scalp stimulation prior to the HSC administration did not result in an enhancement in growth from that of the serum-free HSC administration alone. In order to analyze the distance over which HSC demonstrated efficacy in relation to the injection site, the distribution of hair density in the treated area was averaged over 48 quadrants. The distribution plot suggested that HSC’s effect on hair growth was concentrated within 1-2 mm of the site of injection and at a point central to all 4 injection sites. These results are consistent with publications noting a limited diffusion of Wnt and somewhat greater diffusion of follistatin. These results clearly demonstrate that a single intradermal administration of 10X HSC significantly improved hair growth in subjects with androgenetic alopecia.
This first-in-man clinical trial demonstrates the safety of the cell-secreted HSC protein preparation and is an initial indication of efficacy in hair restoration. The HSC proteins produced by the skin cells grown under hypoxic conditions include factors that are important in hair cycle regulation. The mechanisms responsible for hair growth have been studied for decades as researchers have demonstrated the underlying importance of Wnt proteins and wound growth factors in stimulating dermal papilla-associated stem cells8. Wnt proteins play a crucial role in the later stages of telogen in the initiation of hair growth ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":8981,"position":1}]} 2,9,10 , activating cells and gene expression required for the formation of a hair germ that will commence the next anagen ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":13515}]} 11. Therefore, it is hypothesized that the stimulation of hair regrowth induced by the delivery of HSC is due, at least in part, to Wnt activity. Concurrently follistatin relieves the inhibitory action of BMP2 and activin on hair follicle stem cell proliferation. Wound healing associated growth factors, KGF and VEGF, stimulate keratinocyte proliferation in the developing hair follicle and local angiogenesis to promote hair development.
In order to assess the relationship of the injection site to the growth of new hairs the distribution of hair density was averaged over 48 quadrants from 12 treated patients. Analysis of the distribution of stimulation suggested that stimulation was seen within 1-2 mm of the site of injection (Figure 5), which is consistent with the literature showing limited diffusion of Wnt and the more extensive diffusion of follistatin.
Figure 5. Analysis of the distribution of stimulation suggested that stimulation was seen within 1-2 mm of the site of injection.
Discussion
The efficacy results seen with a single injection of HSC represent a novel approach in hair growth treatment. FDA approved products, minoxidil (e.g. , Rogaine) and finasteride (e.g., Propecia), require daily use of the therapeutic to induce and maintain efficacy ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":16291}]} 12, ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":15547}]} 13, ADDIN ZOTERO_ITEM {“sort”:true,”citationItems”:[{"itemID":11376}]} 14,15. Specifically, these products show their greatest efficacy in reducing loss of hair, with a small percentage of new hair growth seen after at least four months of daily use. In contrast, a single injection of HSC resulted in a statistically significant growth of new terminal hairs and an increase in hair density and thickness at 12 weeks that was still detectable after 1 year. In addition, the hair growth was limited to the region surrounding the injection, suggesting that HSC may provide long term and site-controlled efficacy. This clinical pilot study is the first demonstration that a preparation containing Wnt proteins and growth factors has a biologically active stimulatory effect on new hair induction in humans.
Acknowledgements
We should like to thank Dr. Lior Rosenberg-Belmaker, Dr. David Whiting, Dr. Rolf Hoffmann, Dr. Melissa Carpenter and Dr. Nelly Paz for experimental data, interpretation of pathology, organization of the clinical trial and advice.
References
ADDIN ZOTERO_BIBL 1. Kishimoto, J. , Burgeson, R.E. & Morgan, B.A. Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev 14, 1181-1185(2000).
2. Greco, V. et al. A Two-Step Mechanism for Stem Cell Activation during Hair Regeneration.Cell Stem Cell 4, 155–169(2009).
3. Ito, M. et al. Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding. Nature 447, 316-320(2007).
4. Nakamura, M. et al. Control of pelage hair follicle development and cycling by complex interactions between follistatin and activin. FASEB J 17, 497-499(2003).
5. Maurer, M. , Handjiski, B. & Paus, R. Hair growth modulation by topical immunophilin ligands: induction of anagen, inhibition of massive catagen development, and relative protection from chemotherapy-induced alopecia. Am. J. Pathol 150, 1433-1441(1997).
6. Plikus, M.V. et al. Cyclic dermal BMP signalling regulates stem cell activation during hair regeneration. Nature 451, 340-344(2008).
7. Kashour, T. et al. Myogenic signaling by lithium in cardiomyoblasts is Akt independent but requires activation of the beta-catenin-Tcf/Lef pathway. J. Mol. Cell. Cardiol 35, 937-951(2003).
8. Lowry, W.E. et al. Defining the impact of beta-catenin/Tcf transactivation on epithelial stem cells. Genes Dev 19, 1596-1611(2005).
9. Alonso, L. & Fuchs, E. Stem cells in the skin: waste not, Wnt not. Genes Dev 17, 1189-1200(2003).
10. Andl, T. et al. WNT signals are required for the initiation of hair follicle development.Dev. Cell 2, 643-653(2002).
11. Van Mater, D. et al. Transient activation of beta -catenin signaling in cutaneous keratinocytes is sufficient to trigger the active growth phase of the hair cycle in mice. Genes Dev 17, 1219-1224(2003).
12. Olsen, E.A. & Weiner, M.S. Topical minoxidil in male pattern baldness: effects of discontinuation of treatment. J. Am. Acad. Dermatol 17, 97-101(1987).
13. Price, V.H. , Menefee, E. & Strauss, P.C. Changes in hair weight and hair count in men with androgenetic alopecia, after application of 5% and 2% topical minoxidil, placebo, or no treatment. J. Am. Acad. Dermatol 41, 717-721(1999).
14. Tosti, A. , Iorizzo, M. & Vincenzi, C. Finasteride treatment may not prevent telogen effluvium after minoxidil withdrawal. Arch Dermatol 139, 1221-1222(2003).
15. Long-term (5-year) multinational experience with finasteride 1 mg in the treatment of men with androgenetic alopecia. Eur J Dermatol 12, 38-49(2002).
Follow us: