The perception and manifestation of aging and beauty vary depending on ethnicity and region.1 Recent studies of women having various skin types showed significant differences in the appearance of pores,2 wrinkles and sagging,3–5 as well as transepidermal water loss,6 skin capacitance6, 7 and biochemical markers.8 Furthermore, differences in skin architecture, structure and composition depending on ethnicity can lead to different onsets of aging,9 although UV irradiation is still the most important factor. More recently, the term skin aging exposome was also penned10 to include other environmental factors such as air pollution, smoking or lifestyle.
Concerning visual signs of skin aging, wrinkles are the most important to consumers.11 Facial wrinkles can be grouped together, e.g., as forehead wrinkles, crow’s feet near the eyes, mouth frown lines, the nasolabial folds and vertical lines on the upper and lower lips. In relation, a few studies have been conducted to determine the efficacy of treatments on these disparate facial sites.12, 13
One of the main molecular signs of skin aging is the decrease and disorganization of functional collagen in the dermis.14 This results from either increased collagen degradation, due to chronological aging or photoaging, or from decreased de novo synthesis. Means to protect collagen degradation or to enhance collagen synthesis and maturation would hence be beneficial to counteract skin aging and wrinkle formation. In relation, lysyl hydroxylases (LHs), particularly LH3, encoded by the gene PLOD3,15 and lysyl oxidases (LOXs), particularly LOXL2,16 have been found to be instrumental in the proper maturation of collagen fibers.
Dipeptide diaminobutyroyl benzylamide diacetate, hereafter referred to as DABBAa, is a cosmetic ingredient modeled after a toxic protein found in venom of the temple viper, Tropidolaemus wagleri. The protein, Waglerin-1, acts by inhibiting the nicotinic acetylcholine receptor,17 which blocks muscular contraction. DABBA acts on the same receptor, blocking the muscle nicotinic acetylcholine receptor but in a transient and reversible manner.17 This leads to a relaxation of the mimic muscles in the face, smoothing out facial wrinkles—similar to botulinum toxin but without being injected subcutaneously. In addition, facial expression is not lost.
The present article explores the effects of DABBA by investigating facial wrinkles in three different skin types: Asian, black African and Caucasian. Results indicate both skin type- and facial site-dependent manifestations of the signs of aging. The active also was shown to positively affect the collagen maturation pathway.
Materials and Methods
As stated, DABBA was modeled after a basic amino acid-proline-basic amino acid repeat that is responsible for the activity of Waglerin-1. The repeat consists specifically of arginine-proline-lysine. By distinct side-chain modifications, the peptide Beta-Ala-Pro-Dab-NH-Bzl * 2AcOH was developed for anti-wrinkle activities and tested as described next.
In vitro muscle contraction: Human muscle cells were cultured in gelatin-coated plates until they formed myo-fibers without contraction. To induce contraction, they were co-cultured with spinal cord explants containing dorsal root ganglions from rat embryos. After one day, neurites were growing from the explants. Upon contact with the myotubes, they induced first contractions. After 21 days, the culture displayed high levels of mature neuromuscular junctions with cross-striated, innervated muscle fibers.
At this point, 0.5 mM of DABBA was administered and the cessation of muscle contractions was monitored using an inverted microscope equipped for video recordingb. The results were expressed as the number of fiber contractions in 30 sec, and as a percentage of the contraction frequency compared with the contraction frequency before incubation.
In vitro gene expression in human dermal fibroblasts: Normal human dermal fibroblasts from the abdominal skin of female donors, 18 and 63 years old, were exposed to DABBA for 24 hr. Gene expression averages were measuredc for Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 3 (PLOD3); Lysyl Oxidase Like 2 (LOXL2) was assessed from technical duplicates.
Study 1 in vivo, Caucasian skin: For this full-face, placebo controlled, 28-day study, test creams (see Formula 1) were applied twice daily to the faces of 15 female Caucasians (41–60 years old, 52.5 ± 2). The evolution of wrinkles in their crow’s feet area and foreheads were evaluatedd at baseline and after the four weeks of treatment.
Study 2 in vivo, Caucasian, Asian and black African skin: In a second full-face, placebo controlled, 28-day study, 150 female volunteers having one of three different skin phototypes were enrolled. The test creams (see Formula 1) were applied twice daily. Fifty subjects participated per group, wherein 25 applied a placebo and 25 applied the active test cream.
Caucasians were recruited from Lyon, France (46.2 ± 3.6 years old, Fitzpatrick skin phototypes II–III); Asians were recruited from Bangkok, Thailand (47.3 ± 3.5 years old, Fitzpatrick skin phototypes III–IV); and black Africans were recruited from Quatre Bornes, Island of Mauritius (56.5 ± 4.5 years old, Fitzpatrick skin phototypes V–VI). Cross-polarized images were taken of each subject’s half-face, at a 45-degree angle, and front face, at a zero-degree angle.
Caucasian and black African subjects were photographed using one systeme and Asian subjects, with anotherf. Wrinkle lengths and surfaces were analyzed from the images. To compare the data, measurements were normalized and calculated in mm and mm2.
DABBA blocks muscular contraction, leading to transient muscle relaxation that smooths facial wrinkles.
In vitro Results: Muscle Contraction
In the co-culture assay using human muscle cells and rat spinal cord explants, 0.5 mM of DABBA significantly inhibited muscle contraction by 82% (p < 0.05) in a time-dependent manner 2 hr after application (see Figure 1a). This inhibition persisted for two days and contraction was fully restored after four days, indicating the peptide is fast-acting, long-lasting and fully reversible. These results suggest DABBA correctly mimics Waglerin-1, inhibiting the nicotinic acetylcholine receptor and leading to the relaxation of facial expression lines (see Figure 1b).
To explain DABBA’s anti-wrinkle mechanism of action, consider under normal conditions, acetylcholine (ACh) is released from neurons and binds to the muscle nicotinic acetylcholine receptor (mnAChR). This leads to depolarization and muscle contraction (see left scheme, Figure 1b). On the right scheme (also Figure 1b), DABBA is believed to block mnAChR, making it inaccessible to ACh and in turn, preventing depolarization. Thus, the muscle stays relaxed. It is noteworthy that botulinum toxin acts on the inhibition of ACh release, blocking signaling transfer from neuron to muscle.21 This pre-synaptic activity is slightly different from DABBA, which is acts post-synaptically but exhibits a similar effect.
In vitro Results: Gene Expression
To assess the effects of DABBA on additional anti-aging pathways, human dermal fibroblasts were incubated with the active as described previously. DABBA increased the expression of PLOD3 by 4.5-fold and LOXL2 by 2.4-fold (see Figure 2a). Interesting, for PLOD3, up-regulation was observed only in the 63-year-old fibroblasts, indicating its effects are age-dependent. This may suggest specific activity, where reconstitution of the extracellular matrix is most needed.
To explain DABBA’s effects on the genetic expression of pro-collagen markers, note that both PLOD3 and LOXL2 are involved in the maturation of collagen fibers (see Figure 2b).16, 18 PLOD3 encodes the protein LH3, which adds hydroxyl groups to lysine residues of the pro-collagen molecule. This is essential for pro-collagen to form a triple helix, which is then further processed by LOXL2, among others, to generate mature collagen fibers.22
In vivo Results: Caucasian Skin
As noted, Caucasian female subjects were recruited to assess the activity of DABBA on forehead wrinkles (see Figure 3) and the crow’s feet area (data not shown). Parameters for wrinkle depth, Ra, wrinkle amplitude, Rz, and skin roughness, Rt, all decreased significantly by 15–21% (p < 0.05 for Rt, p < 0.01 for both Ra and Rz) with a formulation containing 4% DABBA; this was not the case for the placebo group.
In vivo Results: Different Ethnicities
In general, it is assumed Caucasian skin shows an earlier onset of the signs of aging than Asian or black African skin.19 This may be due to the decrease in photoaging generally attributed to darker skin phototypes. Recently, this concept was challenged with the finding that aging also depends on the facial site studied.20 This suggests facial aging is a complex phenomenon, dependent not only on skin type, but also on the facial area.
Another study recently found skin hydration shows dramatic variation depending on skin phototype or facial site, with large gradients on relatively small areas.6 These observations inspired us to examine differences in treatment effects of the anti-aging peptide in volunteers having different skin phototypes.
As noted, women from three regions having different skin types applied either a 4% DABBA-containing formula or placebo formula for 28 days. The results in Figure 4a show that the treatment effects were as diverse as the reported appearance of facial wrinkles themselves.
The decrease in forehead wrinkle depth found initially in the Caucasian skin-only study was confirmed (-8.6%, p < 0.05) (see Figure 3) but for the other facial sites, the treatment effects were more complex. For Caucasian skin, a decrease in mouth frown lines and vertical lines on the upper lip was observed, up to 23% in length. Asian subjects showed a decrease in mouth frown lines of 4% in depth, and vertical lines on the upper lip up to 8% for length and depth. Black African skin showed a decrease in the depth of vertical lines on the upper lip by 2%.
A complex pattern of wrinkle appearance was revealed, suggesting facial aging may be more complicated than previously thought.
In addition, the volunteers completed a self-assessment questionnaire (see Figure 4b). Overall, according to the test subjects, the DABBA-containing formula outperformed the placebo formula. Volunteers reported having fewer forehead wrinkles, thus confirming measurements (see Figure 3 and Figure 4a). Furthermore, they felt they had smoother and finer skin, and their facial features seemed lifted.
From the described studies, some conclusions can be made. First, besides its muscle-relaxing activity, DABBA induces the expression of PLOD3 and LOXL2, two genes coding for proteins involved in collagen fiber maturation. This suggests added anti-aging benefits.
Also, in two in vivo studies, one of Caucasian subjects, and the other of subjects having three differently pigmented skin types, significant effects were observed. A robust decrease in the depth of forehead wrinkles and crow’s feet was shown in Caucasian skin. Also, distinct effects on mouth frown lines and vertical lines of the upper lip were observed in three, i.e., Caucasian, Asian and black African, skin types.
Finally, a complex pattern of wrinkle appearance was revealed in volunteers of all three subject groups. Taken together, this data adds to the knowledge that facial aging may be a more complex phenomenon than previously thought. The present findings also allow product developers to develop specific anti-aging compounds dependent on skin phototype or facial site.
Acknowledgements: This study was funded by DSM Nutritional Products.
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