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* Consumer Specialities Personal Care

Preventing hair loss by balancing the hair cycle, strengthening the hair follicle, and improving scalp health By Dr. M. Pfl aumbaum*, M. Farwick, M. Mentel, T. Köhler

Abstract

The overall impression that people make depends to a signifi cant degree on the appearance of their hair. Aging and conditions such as androgenic alopecia result in hair follicle miniaturization, which shortens the growth phase, resulting in hair loss and the formation of fi ne and lifeless hair.

Healthy hair embedded in a healthy scalp is the key to preventing hair loss.

Scientifi cally well-founded claims have been made that substanti-ate the action of Sphinganine, a skin-identical sphingolipid that tar-gets hair loss by balancing the hair cycle, strengthening the hair follicle, and improving scalp health.

Hair growth occurs over a cycle consisting of growth (anagen), re-gression (catagen), resting (telogen), and exogen phases1, 2) (fi gure 1).

Sphinganine-based ceramides represent the most abundant cera-mide class present in human hair 3). Based on this and other scien-tifi c data, sphinganine seems to be a promising active ingredient for hair-quality promoting cosmetic applications.

Sphinganine is produced from renewable raw materials such as sugar in a sustainable biotechnology process.

The fermentative production process ensures that Sphinganine features the same stereochemical confi guration as found in nature and in human skin (fi gure 2). The skin-identical stereochemistry of Sphinganine is of key importance for its biological functions.

Figure 1: The hair growth cycle

The hair follicle undergoes a repeated growth cycle and is able to regenerate a new hair shaft during each cycle. More than 80% of the hair in young and healthy individuals is actively growing (anagen phase). Aging and other circumstances such as androgenic alopecia result in hair follicle miniaturization, which shortens the anagen phase, resulting in hair loss and the formation of fi ne and lifeless hair.

Healthy hair embedded in a healthy scalp is the key to preventing hair loss.

Figure 2: The chemical structure of Sphinganine. The absolute stereochemistry on the asymmetrical carbon atoms is indicated in brackets (in purple)

Sphinganine prolongs the anagen phase and improves scalp health and scalp renewal capacity, making it an ideal active ingredient for preventing hair loss (fi gure 3a, 3b)

Figure 3b: Proposed working mechanism of Sphinganine withinthe hair follicle and on the scalp

Figure 3a: Proposed working mechanism of Sphinganine within the hair life cycle

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Various in vitro studies have shown that Sphinganine balances the hair cycle by inhibiting 5-�-reductase, by improving the capacity of the scalp to renew itself, by stimulating the formation of essential building blocks such as proteins and ceramides, and by balancing microfl ora to improve scalp health.

Preventing hair loss by inhibiting 5-�-reductase (in vitro)

The aim of this study was to determine the potential of Sphinga-nine as an inhibitor of 5-�-reductase type I, which is the key enzyme catalyzing irreversible reduction of testosterone (T) to dihydrotes-tosterone (DHT), the most potent androgen. DHT has been identi-fi ed as key factor involved in androgenetic male baldness.

The inhibitory potential towards 5-�-reductase type I was tested in a cell-free assay using cell homogenates isolated from stably trans-fected HEK293 cells, yielding an IC50 of 6.6 µM for Sphinganine (fi gure 4). Finasteride, a known 5-�-reductase inhibitor, was included as the internal positive control.

The aim of the study was therefore to determine the global effects of Sphinganine treatment in a confl uence-induced, in vitro keratino-cyte differentiation model, particularly with respect to lipidomic and transcriptomic changes.

Sphinganine treatment strongly promotes ceramide production in keratinocytes (Figure 5).

Figure 4: Inhibition of 5-�-reductase I by Sphinganine in vitro

The test results show that Sphinganine is a potent inhibitor of 5-�-reductase type I. Consequently, Sphinganine is a suitable cos-metic ingredient for inhibiting the conversion of testosterone to di-hydrotestosterone. This will positively infl uence the hair growth cycle by preventing the transition to the catagen phase and follicle miniaturization, particularly among men.

Moreover, reduction of scalp sebum production will lead to a less greasy scalp and an overall improvement in scalp health.

Improvement of the scalp barrier function and renewal capacity (in vitro)

A functional skin barrier is a prerequisite for a healthy scalp. By contrast, compromised barrier function can lead to a dry, itchy scalp. Improving the barrier formation should therefore lead to an improvement in overall scalp health, with the additional, indirect benefi t of combatting hair loss.

Figure 5: Increase in cellular ceramide content after four days [nmol/mg protein]

Overall ceramide pools were stimulated in a balanced way, leading to concomitant upregulation of all major ceramide classes containing sphinganine, phytosphingosine, or sphingosine in their backbones. In particular, very long-chain fatty acid cera-mides, which are deemed critical for intact barrier function, were upregulated. Moreover, microarray analysis (data not shown) in-dicates signifi cant induction of overall sphingolipid metabolism and keratinocyte differentiation caused by the Sphinganine treat-ment.

In addition to applying Sphinganine on differentiating epidermal keratinocytes, a complementary experiment was conducted on a non-differentiating keratinocyte model in order to explore effects on the transcriptome of proliferating epidermal keratinocytes. The purpose of this experiment was to explore the effects of Sphinga-nine once it had penetrated deeper layers of the viable epidermis, the follicle shaft, and the hair root. Because the hair shaft arises from rapidly proliferating keratinocytes in the bulb (one of the highest rates of proliferation in the body), special attention was paid to genes known for their roles in cell proliferation. This class of genes includes growth factors, as well as genes controlling synthesis and turnover of the intracellular messenger molecule sphingosine-1-phosphate. Another class of genes relevant in the context of proper hair structure is one that mediates cell-cell interaction and includes integrin and cadherin genes.

In summary, the gene expression data obtained for undifferenti-ated, proliferating keratinocytes suggest that Sphinganine maintains the proliferative state of keratinocytes and favors epidermal renewal (fi gure 6).

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In particular, the stimulation of basic fi broblast growth factor ex-pression, as well as the stimulation of gene expression related to sphingosine-1-phosphate and sphinganine-1-phosphate synthesis, indicates that treating keratinocytes with Sphinganine triggers sign-aling cascades that ultimately promote cell proliferation and coun-teract apoptosis. Assuming that these signaling cascades are conserved, it seems likely that the positive effects of Sphinganine treatment also apply to hair matrix keratinocytes, which represent one of the fastest proliferating cell populations in the body. As such, Sphinganine will likely keep hair follicles in an active, growing state, and prevent follicles from prematurely undergoing apoptosis and entering the catagen phase. Moreover, developing hair follicles ben-efi t from inducing genes for cadherin and integrin, two classes of proteins that mediate cell-cell and cell-matrix adhesion processes, and that are critically important for proper hair follicle and hair shaft architecture.

Improvement in scalp health by equalizing micro fl ora (in vitro)

Overall scalp health is always the outcome of multiple infl uencing factors. One important factor is scalp microfl ora. While the natural scalp microfl ora comprises many different microbe species, yeast strains belonging to the genus Malassezia have been identifi ed as a major determinant of scalp health. Numerous studies have demon-strated the key role that Malassezia strains play in the context of scalp health, particularly with respect to dandruff and seborrheic dermatitis. Although Malassezia strains are part of normal cutane-ous microfl ora, they are known to be involved in the occurrence and severity of certain human skin conditions, including dandruff, seb-orrheic dermatitis, folliculitis, and others.

Another mechanism contributing to a healthy scalp is mediated via the expression of anti-microbial peptides (AMPs). These peptides act mainly by interfering with the cytoplasmic membrane of target microbe strains, causing membrane leakage and ultimately cell death. One of the most prominent anti-microbial peptides is human �-defensin 2 (HBD2). Expression of AMPs correlates with the ke-ratinocytes’ differentiation status, and together with the mechanical stability conferred by the stratum corneum, AMPs effi ciently help maintain overall skin and scalp health.

Sphinganine improves scalp health directly via antimicrobial ac-tivity and indirectly by stimulating HBD2 formation.

An in vitro germ count reduction test revealed profound direct anti-microbial activity against Malassezia furfur (fi gure 7), which is known to cause dandruff. Moreover, Sphinganine treatment stimulates HBD2 gene expression in an in vitro keratinocyte model (fi gure 8).

Figure 6: Stimulation of proliferation markers (gene expression after 24h)

Figure 7: Malassezia furfur germ count reduction. 1% Sphinganine efficiently reduces M. furfur germ counts by three orders of magnitude within 20 minutes.

Figure 8: Effect of Sphinganine on human �-defensin 2 (HBD2) gene expression in primary human keratinocytes after 72h of incubation (solvent: 0.1% DMSO).

The profound stimulation of HBD2 gene expression refl ects Sphinganine’s potential to stimulate innate immunity via produc-tion of anti-microbial peptides (AMPs). As such, Sphinganine likely strengthens the skin’s defenses and ultimately contributes to overall skin and scalp health.

Extended in vivo studies have confi rmed that Sphinganine signifi -cantly reduces hair loss by prolonging the anagen growth phase of hair and effectively improves hair quality and scalp health.

Improvements in hair quality and scalp health (in vivo)

The aim of the study was to investigate the potential effect of Sphinganine on prolonging the hair anagen phase and improving overall hair quality and scalp health.

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The TrichoScan® method was employed to objectively determine state of the hair life cycle by determining the anagen and telogen rate. Visible and otherwise detectable effects were assessed by ex-pert rating and photographic documentation.

An ethanolic hair tonic containing 0.1 – 0.5% Sphinganine was provided, and volunteers evenly applied this to their scalps (dry hair) and hairlines in the morning and evening, aided by some gentle massage. Measurements were conducted before the fi rst application and after 8 and 16 weeks of product application. Data were consid-ered from a total of 96 test subjects.

The TrichoScan® images clearly show the effects of Sphinganine application (fi gure 9). The increase in anagen hair leads to a notice-able increase in the amount of hair on the scalp. The TrichoScan®

images also indicated benefi cial effects on scalp health.

groups, the anagen rate increased, and the increase was highest in the 0.5% Sphinganine group (2.7% and 2.8% after 8 and 16 weeks, respectively). In the 0.2% and 0.1% Sphinganine groups, increases of 1.8%/1.9% and 1.1%/1.1% were determined (after 8/16 weeks), re-spectively.

Relative to vehicle control, the increase in the anagen rate in the 0.5% Sphinganine group was 4.3%/3.9% (after 8 and 16 weeks, re-spectively), 3.3%/3.0% in the 0.2% Sphinganine group and 2.7%/2.3% in the 0.1% Sphinganine group.

In terms of the expert rating (fi gure 12), all hair quality and scalp health parameters improved after application of Sphinganine, re-gardless of the concentration. In particular, hair quality parameters were greatly improved in all Sphinganine groups compared to vehi-cle control, and improvements in hair volume scored best.

Figure 9: Sample photographic images recorded using the TrichoScan® method for two male volunteers from the Sphinganine group, before application (left column) and after 16 weeks (right column). Improvements in anagen hair and scalp health are clearly visible.

Photographic documentation was standardized, hair was washed the day before measurement, and no styling products were used. Subjects did not use any hair tonic on the day of visit.

The photographs (fi gure 10) clearly show the effect of Sphinga-nine application, and a noticeable increase in the amount of hair due to improvements in hair anagen rate. The effects were achieved after applying as little as 0.1% Sphinganine.

TrichoScan® images were analyzed as well. The difference be-tween the growing (anagen) and non-growing (telogen) hair can be seen. The recorded images were used as a basis for calculating the anagen hair rate [%] (fi gure 11).

In the vehicle control group, the anagen rate decreased by 1.6% and 1.1% after 8 and 16 weeks, respectively. In all Sphinganine

Figure 10: Photographic documentation of the effect of sphinganine application on two male study participants.

Figure 11: Anagen rate as determined by TrichoScan® measurements. An increase in the anagen rate was detected that was particularly pronounced when compared to vehicle control application.

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Figure 12: Changes in hair quality and scalp health parameters as rated on a five-point scale by experts on the basis of a questionnaire.

Conclusion

Overall, SPHINGONY (trade name for Sphinganine) is a naturally occurring, skin-identical molecule particularly targeting male pat-tern hair loss by balancing the hair cycle, strengthening the hair follicle, and improving scalp health. SPHINGONY is COSMOS certi-fi ed and approved by Ecocert Greenlife according to the Ecocert Standard for Natural and Organic Cosmetics, available at http://cos-metics.ecocert.com.

References

1. Driskell et al., J Cell Sci 2011, 124, 1179 – 1182

2. K.S. Stenn, R. Paus; Physiol Rev 2001, 81, 449 – 494

3. G. Hussler, G. Kaba, A.M. Francois, D. Saint-Leger, Int. J. Cosmetic Sci. 1995, 17, 197 – 206) ■