Enzymes for Skin Protection: COSSMA

photo: Yuganov Konstantin/Shutterstock.com

Skin ageing can be promoted by many factors. Some require fast and long-lasting countermeasures in order to effectively protect the skin from the visible signs of premature ageing. Enzymes offer the ideal preconditions for this.

Skin protection is not a question of age. Every day, our skin is exposed to many factors that promote premature skin ageing. Two very important factors are closely related to each other: UV radiation and free radicals. UV radiation alone is responsible for 80 percent of the visible signs of facial skin ageing1. UV radiation and free radicals can lead to ageing signs like photoageing and wrinkles through DNA damage and oxidation processes. Therefore, our skin needs a reliable repair and protection system.

Numerous active ingredients are known, especially antioxidants for free radical neutralisation. However, in DNA repair and the neutralisation of free radicals, effectiveness alone is not the decisive factor. Speed and a long-term effect are of high importance. The search for suitable active components took several years of research. A crucial path led to the mechanisms of nature, which have proven their effectiveness for billions of years.

Active ingredient complex

Following nature’s example, enzymes are ideal components. They enable and accelerate many biochemical reactions and have important functions in the metabolism of organisms. A key feature is that enzymes are not used up, even over many reaction cycles, and therefore uniquely combine efficiency and long-lasting effect. Thereby, enzymes can rightly be called conductors of life.

Our new active ingredient² for holistic skin protection mainly contains a synergistically acting complex consisting of two enzymes: the repair enzyme photolyase (derived from microalgae) and an antioxidant enzyme (iron peptide). Liposomal encapsulation of the two enzymes additionally improves skin penetration.

Repair enzyme photolyase

Cyclobutane Pyrimidine Dimers (CPDs) are the most common UV-induced lesion of DNA: UV radiation separates opposite base pairings in the DNA double helix and two adjacent bases of the same strand subsequently combine incorrectly.

Photolyase repairs the so-called CPDs very efficiently and 10 - 100 times faster than the body’s own repair mechanism, so that this important system can be optimally supported. In the latter, the DNA is repaired via an intermediate in which the CPDs are generously separated and the DNA strand is subsequently re-synthesised. Photolyase cleaves the CPDs directly in only one step.

Antioxidant enzyme

The antioxidant enzyme (iron peptide) was developed to continuously neutralise Reactive Oxygen Species (ROS), also including free radicals. Through self-regeneration, it is not used up as it is an enzyme. This offers long-term radical protection until it is naturally degraded by skin-physiological metabolic processes.

The effect of free radicals is, simplified, the drive for paired electrons. Free radicals have unpaired electrons (electron gap). They compensate for this electron gap by taking electrons from other molecules (for example components of skin cells). This creates an electron gap in these molecules, which corresponds to a damage.

Antioxidants have flexible electrons that they can donate to free radicals (neutralisation). Antioxidants can balance the resulting electron gap well, for example through their structure. The other molecule remains unaffected and no damage occurs. However, antioxidants are used up in this state and can no longer neutralise further free radicals. Their protective effect is temporary. Consequently, damage can occur to cell components. The antioxidant enzyme targets precisely this point: it neutralises free radicals like ordinary antioxidants, but is subsequently able to regenerate itself.

Like an enzyme, it does not consume itself and can therefore continuously neutralise many more free radicals. Through this long-term radical protection, skin cell components remain protected from oxidative damage for a longer period of time.

The illustration shows repair mechanisms for the removal of Cyclobutane Pyrimidine Dimers (CPDs).

Methods and results

To study the DNA repair, human 3D full thickness skin models3 were used. The diagram above shows a significant reduction of UV-induced CPDs as well as a synergistic effect of the two enzymes (increased reduction of CPDs).

The long-term radical protection was analysed with hydrogen peroxide (H2O2) solution as an initiator for the generation of free radicals respectively ROS.

The treatment with H2O2 was repeated every 24 hours with fresh solution over an examination period of 96 hours. Within 24 hours after application of the active ingredient solution and initial treatment with H2O2 solution, ROS could be reduced by over 76 percent and after 48 hours and second treatment with fresh H2O2 solution even by more than 80 percent.

The reduction of ROS can be detected significantly even after 72 and 96 hours accordingly, whereby the degree of reduction decreases with time, as the antioxidant enzyme is degraded over this long period of time. As in the CPD reduction study, a synergistic effect of the two enzymes was demonstrated.

Discussion

The study results show a highly efficient DNA repair as well as a long-term radical protection and furthermore a synergistic effect. This bidirectional synergism represents a significant performance advantage compared to the single enzymes. The respective enzyme concentrations were also optimally harmonised with each other in several screening studies. Since for both effects the skin would first have to be damaged in order to be able to demonstrate the subsequent repair of the DNA and the reduction of free radicals, no in vivo studies were deliberately carried out for ethical reasons. Thus, human 3D full thickness skin models were chosen for the CPD reduction studies.

These skin models also have the advantage that they are very robust (feasible study designs) and provide high reproducibility4. Human keratinocytes cell cultures have proven to be best suited for the extreme conditions of the studies on the reduction of free radicals. Therefore, the degree of damage could be widely exploited to investigate the potential of the active complex with repeated applications of a H2O2 solution over an examination period of 96 hours. However, in vivo studies are currently being carried out to prove the resulting effects, such as wrinkle depth reduction and moisture retention, in order to complement the proof of concept already presented.

Study design: human 3D full thickness skin models, used formulation: aqueous solution with 1 percent active ingredient or only with the corresponding photolyase concentration. Subsequent irradiation: UVB radiation  (220 mJ/cm2), incubation for 24 hours. Untreated,  UV-irradiated = positive control (normalisation to 100 percent, maximum stress); untreated, not irradiated = negative control. Analysis: CPD ELISA assay (epidermal keratinocytes), percent values in relation to positive control (p<0.01).

Conclusion

UV radiation and free radicals are two key factors, which are primarily responsible for premature skin ageing. Enzymes are optimal active components to counteract premature skin ageing. They provide a fast and a long-term effect – essential requirements for efficient repair of UV-damaged DNA and neutralisation of free radicals.The repair enzyme photolyase from microalgae in combination with an antioxidant enzyme (iron peptide) in liposomal encapsulation mainly forms the synergistic active complex described herein.

It provides holistic skin protection like an invisible shield to protect the skin from the negative effects of UV radiation and free radicals. Due to the enzymatic action, a long-term effect is also possible through continuous repair of DNA damage and long-lasting neutralisation of free radicals, which significantly exceeds the duration of action of common antioxidants.

References

Flament, F., et al., Effect of the sun on visible clinical signs of aging in Caucasian skin, Clinical, Cosmetic and Investigational Dermatology 2013, 6, 221-232.
Glorydermal Guard
Mewes, K. R., et al., Reconstructed 3D Tissues for Efficacy and Safety Testing of Cosmetic Ingredients, IFSCC Magazine 2017, 20 (2), 55-64.
Reisinger, K., et al., Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility, Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2018, 827, 27–41.