In the early 80s researchers observed that during the natural process of aging in both animals and humans, total protein content in several tissues decreases1. The main cause of this reduced protein content, it has been suggested, is related to slower cellular metabolism with decreased protein synthesis and a parallel increase in protein degradation1, 2. In skin fibroblasts from rats and humans, and also in vivo, the rate of protein synthesis decreases with age, with proteins such as collagen being a marker of reduced synthesis-associated metabolism2, 3, 4. The decrease in skin protein content during aging is not only due to a slower metabolism and reduced synthesis, but also to increased protein degradation by metalloproteases, such as MMP1 and MMP3, with a parallel decrease in TIMP1, a tissue inhibitor of metalloproteases2.
Changes in overall skin protein content parallel a decrease in proteins that are important in guaranteeing normal skin turnover. These include proteins such as the cytokeratins, involved in the cornification process associated with keratinocyte differentiation and maturation5, as well as proteins important in the maintenance of the skin’s structural architecture, such as filaggrins in keratinocytes6 or collagens and elastin in fibroblasts2, 3, 4. The lack or decrease of these proteins causes major changes in skin structure and overall functionality. Typically as skin ages, these changes translate at the macroscopic level into thinner and more fragile skin with a slower cornification process. The skin loses its elasticity and radiance and starts to develop fine lines and wrinkles.
Based on the hypothesis that the stimulation of protein synthesis ameliorates skin roughness and wrinkle profile in aged skin, we have formulated a cosmetic complex called Uniprosyn PS-18. This complex is a carefully balanced blend of a protein biosynthesis precursor, hydrolyzed oat protein extract, which is rich in amino acids, combined with energy boosters adenosine triphosphate (ATP) and niacinamide, both involved in the formation of the NAD+/NADH redox complex (INCI in Figure 1).
What is particularly interesting is that 29% of the hydrolyzed oat protein extract is composed of glutamine and glutamic acid. These two amino acids are well known precursors for protein synthesis7, 8, 9, 10. Moreover, the intermediate glutamate is not only a precursor for protein synthesis9, but has signaling functions binding to the N-methyl-D-aspartate (NMDA) and family related receptors on the membrane of keratinocytes. This triggers the synthesis of differentiation markers such as glutamase, cytokeratins and filaggrin, proteins important for normal cell turnover11-17. Therefore, the presence of this extract containing high amounts of glutamine and glutamic acid would strongly stimulate protein synthesis in the skin and decrease visible signs of aging in vivo. Adding niacinamide and ATP could further boosts enzymatic activities related to protein biosynthesis, decreasing the effect of aging as previously shown in vitro in primary human fibroblasts18, 19 and in vivo on human volunteers where face wrinkles and skin elasticity were measured20. The proposed mechanism of action of Uniprosyn PS-18 is described in Figure 2.
Glutamine enters through the cell membranes of keratinocytes and acts directly as a precursor for protein synthesis. Glutamine can also be converted into glutamate by the enzyme glutaminase and glutamate can convert back to glutamine by using the enzyme glutamine synthetase. Glutamate can also act as a precursor for amino acids and eventually protein synthesis, but could also be secreted in special conditions from the cell, conditions that require membrane depolarization (associated with ion flux), and that are associated with the need for novel protein synthesis17. The secreted glutamate is then captured by a transporter protein and released to bind to the NMDA family of receptors. A subsequent Ca++ uptake by the receptor then activates a signaling cascade leading to the production of differentiation markers such as glutamase (that would further convert glutamine), filaggrin and cytokeratins. Niacinamide and ATP enter the cells and stimulate further production of NAD+ that, together with its reduced form NADH, boosts enzymatic reactions associated with protein synthesis.
To test the efficacy of Uniprosyn PS-18 in stimulating protein synthesis we incubated it at 1% with reconstituted human skin followed by total protein measurement (details in Materials and methods). To verify its efficacy in vivo, we formulated Uniprosyn PS-18 in a cream at 3% and applied it to the skin of human volunteers for 56 days and then measured skin roughness and wrinkle depth (details in Materials and methods).
Materials and methods
Studies in reconstituted human skin – measurement of protein synthesis
To evaluate the effect of Uniprosyn PS-18 on protein neo-synthesis, a standard assay which measures the incorporation of 3H-leucine was used.
Uniprosyn PS-18 at 1% in water was applied topically to the reconstituted human skin epidermis (SkinEthics, France; 50 µl/epidermis). Three epidermises were left untreated (control). The treatment was performed in triplicate. The epidermises were cultivated in SkinEthic medium in the presence of 50 µCi/ml (final) of L-[4, 5-3H]-leucine (Amersham TRK510 (5.99 Tbq/mmol, 162 Ci/mmol)) at 37 °C in an atmosphere of 5% CO2. After 72 hours of treatment, the proteins were extracted from the epidermises using a chaotropic buffer (Tris/Hcl 50 mM, guanidine 4 M and EDTA 5 mM, pH 8.0), then precipitated using trichloracetic acid (TCA), filter collected (collector and filters Skatron), washed with cycles of TCA and ethanol 70%. Finally the incorporation of 3H-leucine was measured by liquid scintillation counts (cpm).
Studies on human volunteers – experimental set up
Twenty-five subjects were selected, between 37-65 years of age (average: 44.6). Subjects were instructed not to use any topical preparations on the test areas starting seven days prior to testing until the end of the test. For cleansing, only water or a mild syndet (Eubos® flüssig – blau; manufacturer: Dr. Hobein, D-53340 Meckenheim- Merl, Germany) was allowed (whole study including the run-in phase).
Prior to the first application of a cream containing Uniprosyn PS-18 at 3% or the placebo cream, measurements were taken at clearly defined sites on the inner sides of the forearms and in the crow’s feet region around the eye. One area on the inner side of the forearm remained untreated and served as a control throughout the study, while one area was treated with the cream containing Uniprosyn at 3% and another area with the placebo cream. When the study was performed in the crow’s feet region around the eye, one side of the face was treated with the cream containing Uniprosyn at 3% and the other side with the placebo cream. Further measurements were performed after 14, 28, and 56 days of application, 8-12 hours after the last daily application (adaptation time: 30 min, room temperature: 21±1°C, relative humidity: 50± 5%). The subjects used the products (approximately 2 mg/cm2) twice daily (in the morning and evening) in home application after a demonstration in the test institute in a manner corresponding as closely as possible to that to be used by future consumers.
Skin roughness and wrinkle depth measurement
PRIMOS technology was used21 to measure skin roughness and wrinkle depth.
PRIMOS (phase-shifting rapid in vivo measurement of skin) is a non-contact measurement device that allows for real-time three-dimensional in vivo measurement of the microtopography of human skin based on the technology of active image triangulation. The measurement head consists of a digital micromirror device as the projection unit and a CCD-camera as the recording unit, mounted onto an adjustable rack. For active image triangulation, an intensity encoded point M is projected onto the surface under investigation. Its image on the surface is recorded by the CCD-camera from a specific angle. The point M is a function of parameters like intensity, triangulation angle between the projection system and the camera, and some other coordinates of the camera and projection plane. The height information of the structured surface is coded into the distorted intensity pattern and recorded. The resolution and accuracy depend on the optical and topographical characteristics of the measured surface and on the noise characteristics of the measurement system. For accurate in vivo measurements of human skin, depending on the measured part of the human body (inner forearm, forehead, eye zone), different parameters of effective wavelength and amplification factor are used. To study the differences in human skin and avoid undesired distortions by movements, the fast phase-shift technique was used for the measurement. Distortions in the topography due to hairs can be digitally removed by the PRIMOS software.
Skin roughness was assessed by means of the parameter RZ (mean depth of roughness). To mitigate potential directional effects, the evaluation was conducted using the arithmetic average of RZ from 32 radial cuts.
The mean depth of roughness is defined as: