Entrevista originalmente publicada em/ Originally published by: https://www.fightaging.org/

OneSkin Technologies is one of the few companies in the present community of startups focused on rejuvenation and slowing aging to adopt a serious cosmetics focus on development. Here "cosmetics" is a regulatory term, not an indication of something used for the purposes of looks: it is perfectly possible for a topically applied product that is regulated as a cosmetic to have therapeutic effects, just like a drug. Nonetheless, cosmetics and drugs have entirely distinct paths of regulation, very different from one another, and each with their own costs and challenges. In regulated cosmetics development there is no animal testing at all, everything proceeds to human trials on the basis of tissue models of skin. The trials themselves are quite different. It is arguably easier to run a rejuvenation therapy through the cosmetics regulatory pathway than to try to introduce it as a drug, provided that has a significant effect on skin aging.

This is the direction taken by OneSkin, where the staff are working on a line of senolytic compounds to selectively destroy senescent cells in aged tissues, and that will be developed as cosmetic products at the outset. I met the OneSkin founder earlier this year, and had a chance to pose a few questions about the work being carried out at the company. I think that this approach to the challenge of medical development is worth watching, particularly given that the next major area of rejuvenation research to take off may be cross-link breaking. Cross-links are influential in the age-related loss of elasticity in tissues such as skin and blood vessels. I imagine that companies analogous to OneSkin will emerge quite quickly in that space, once it has reached the same level of maturity as presently exists for senolytics research and development.

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How did OneSkin Technologies come about? What led you into cosmetic senolytics?

OneSkin's initial proposal was to validate the effectiveness of "anti-aging" skincare products available in the market, in order to meet the needs of consumers for science-validated products as well for the companies that are looking to differentiate their products from competitors. Our approach for this validation was to test a given molecule in 3D human skin equivalents and analyze changes in the methylation pattern by running age-predictor algorithms, such as the Molecular Clock developed by Steve Horvath in 2013. Since this and other algorithms used at the time largely failed to predict skin age accurately, we decided to develop our own skin-specific molecular clock, in which the average difference between predicted age and chronological age is lower (approximately 4.6 years) than the currently available molecular clocks. Later on, we realized that we could create more value and offer a scalable solution by developing new and more effective products for skin rejuvenation, instead of limiting ourselves to validating third party products. We also realized that there wasn't any initiative for targeting senescent cells focused on our body's largest organ, the skin.

We believe the skin will be the first tissue to benefit from a senotherapeutic approach since it allows for topical application, virtually no contact with the bloodstream, and possibly a faster route to the market, if categorized as a cosmetic. We also love the proposal to develop senotherapeutics for skin because their effects will be visually perceived by consumers. Finally, the International League of Dermatological Societies (ILDS), a global, not-for-profit organization representing 157 dermatological societies worldwide, has identified the consequences of skin aging as one of the most important grand challenges in global skin health. Reduced functional capacity and increased susceptibility of the skin with development of dermatoses such as dry skin, itching, ulcers, dyspigmentation, wrinkles, fungal infections, as well as benign and malignant tumors are the most common skin conditions in aged populations worldwide and may be prevented with the use of technologies that have been designed to promote skin age reversal, like ours.

The audience here is more familiar with the FDA process for new drugs. How does cosmetics development differ from that?

Here in the US, the law does not require cosmetic products to have FDA approval before they go on the market, but there are laws and regulations that apply to cosmetics on the market, including the voluntary cosmetic registration program. Despite the general feeling that cosmetics are hardly regulated by FDA, safety is the number one rule, accompanied by the important observation that cosmetic products must be properly labeled. This means that any cosmetic product in the market should do no harm to the skin. For this purpose, there are guidelines to be followed when introducing a new molecule in a cosmetic product.

Basically, the company should provide data regarding mutagenesis and chromosomal changes by performing tests such as the Ames test (which uses bacteria to analyze the potential of a given compound to cause DNA mutations), cytotoxicity (using human cells) and karyotyping analysis (using human cells). Since OneSkin does not use animals to develop products, additional safety studies using the complete formulation to assess skin irritation, corrosion, and sensitization are performed in human skin equivalents (in vitro) and also in human subjects. Additional tests, such as ocular toxicity are desirable and even mandatory according to the cosmetic product. At OneSkin, we performed most of the cited tests, including cytotoxicity in human fibroblasts and keratinocytes derived from different donors, mutagenesis (Ames) and chromosomal aberration (karyotyping), toxicity through human skin equivalents, and, finally, we have already performed the Repeated Insult Patch Testing (RIPT) in 54 human subjects, provided by an independent contract organization. All of them came out clear and in accordance with the parameters required, guaranteeing safety for our future clients.

Tell us something about your development. What is your candidate molecule, and how far along are you in the process of validation leading to human use?

Our lead candidate molecule is a new synthetic peptide, which was initially screened in a synthetic library for antimicrobial peptides (AMP). AMPs have multifunctional behavior and accumulate several interesting properties for skin applications, including tissue repair, antioxidantactivity, collagen synthesis, anti-inflammatory activity and we decided to evaluate their senotherapeutic potential. From our initial 200 library, we selected 4 hits - the 4 compounds which were most effective in decreasing senescent cells in human skin. Then, we used an algorithm to create variations of such sequences, leading to hundreds of possible leads. Among those leads, we selected the two best peptides, deemed OS-1 and OS-2, which have consistently shown the ability to decrease human cellular senescence caused by aging, ultraviolet light, and other types of genotoxic stress by 25-50%.

It is worthy of mention that we chose to build a human cell-based platform in order to close the gap between preclinical and clinical scenarios and to mimic skin aging as closely as possible. Indeed, our valuable technological platform is proprietary and has been patented. To briefly outline our pipeline, first, we evaluate the ability of new compounds to decrease cellular senescence through two markers. The classical senescence associated-β-galactosidase (SA-β-Gal) marker is analyzed, as it has been consistently used in the aging field for a least 30 years and is considered an easily identifiable marker of cellular senescence. Nevertheless, since SA-β-Gal also has important limitations, we complement our analysis with a more recent and sensitive marker of cellular senescence, ATRX foci formation.

For each compound, we test both markers in cells obtained from at least three different healthy and aged donors. As positive controls, we have used senolytic and senomorphic molecules, such as fisetin and rapamycin. We also have tested most of senolytics described in the literature and most have failed to induce apoptosis in senescent cells in those cell types or show nonspecific effects, causing a significant toxicity to non-senescent cells. To date, our peptides are the molecules that have performed the best, considering safety and efficacy endpoints. We have been able to replicate human skin aging in vitro by growing skins with cell donors of diverse ages, ranging from a neonatal (0Y), to young (approximately 30Y) to aged (over 50Y). We have characterized these models according to skin equivalent structural organization, gene expression, and accumulation of senescent cells. Using aged skin equivalents, we test compounds by adding them into the culture media for 5 days. At this time, histological, SA-β-gal staining and qPCRanalysis are performed to evaluate the skin health and the senotherapeutic and age reversal potential of such molecules. Additionally, we have formulated our main peptide in a topical cream and have applied onto skin biopsies of aged donors, and we could observe an improvement in epidermal thickness after 5 days of treatment.

Importantly, OS-1 is performing better than retinoic acid, currently considered the gold standard molecule for anti-aging skincare products. It is also worth mentioning that we have consistently seen an increased expression of p16 and inflammatory cytokines like IL-6 and IL-8, along with the "peeling effect" of retinoic acid, which is usually perceived in human use.

Finally, after performing these in vitro studies and clearing the safety (including an IRB approval) of our lead candidate, we have improved our own topical OS-1 formulation and began testing it on a group of 23 healthy volunteers, ranging from 32 to 84 years old. This experiment was initially focused on safety assessment only, but we already consider it a first validation of OS-1 effectiveness in humans, since our preliminary data is extremely promising, with 100% safety and a significant visual improvement observed in most patients within the first month of continuous use. Later this year, we will proceed to a randomized, placebo-controlled clinical study provided by an independent contractor organization.

Why can't one just use dasatinib, or other established senolytics, in some form and spread it on skin? Why something new?

Despite major proofs of concepts generated in the longevity field recently, the clinical use of senolytics must be carefully evaluated. We initially tested several senolytics like dasatinib and unfortunately, the majority that we tested on our platform were either very toxic even to non-senescent cells, or are not effective in decreasing the relative percentage of cellular senescence. Initially, we were disappointed by the lack of reproducibility in our hands, but since we have tested such compounds consistently, we believe that the discrepancies observed may result from different experimental settings, since most papers are based on animal models or genetically-modified cell lines, while we work with healthy and normal aged human cells.

Furthermore, when tested in skin equivalent models, senolytics continued to be highly toxic, compromising skin equivalent general structure, and decreasing the thickness of the epidermis. ABT-263, A1331852 and Dasatinib + Quercetin are but a few senolytics we have already tested. We have tested other molecules which are safer and effective like fisetin, but this molecule has the limitation of requiring high concentration to be effective (i.e. 20 μM), due to its low bioavailability. Fisetin's natural color (yellow to orange) also impairs its use in a topical product.

If your product works topically on skin, why not administer it systemically to clear out senescent cells throughout the body?

As mentioned before, we believe skin health is an important and highly overlooked target to rapidly promote drastic improvement of wellness, self-confidence, and prevention of aging-related skin disorders. Therefore, we have chosen skin as a starting point to validate our lead molecule and start bringing its benefits earlier to consumers. Once OS-1 ́is proven to be well-tolerated and effective at reducing skin senescence when applied topically, it will then open several avenues to explore other indications of the peptide throughout the body, which would fall in the regular FDA pathway for drug development. In this regard, one basic assay we performed in order to assess the potential application of OS-1 for longevity purposes was the evaluation of healthspan improvement and lifespan extension in C. elegans worms. In this experiment, parameters of healthspan (thrashing and esophageal pumping - which basically indicates how well the worm moves and eats) were improved, and the treated worms lived longer (median life extension increased approximately 12%).

This is comparable to other age reversal strategies published in the literature, and reflects the potential of OS-1 to be used for other therapeutic applications in the future. The positive result surprised us, because we have not yet optimized our candidates by applying any medicinal chemistry. However, this will soon be performed once the mechanism of action is elucidated. On this subject, we have shown that OS-1 promotes a decrease of cellular senescence levels of cells and tissues by promoting apoptosis (decrease in phosphorylated Akt on Ser473), increasing DNA repair capacity (induction of BLM and SIRT6 gene expression) and preventing DNA-damage induced senescence (UVB exposure induction model).

What other areas of rejuvenation research do you think would benefit from a cosmetics approach, to speed adoption?

Most strategies targeting one of the hallmarks of aging could be useful for a cosmetic approach. The main limitation we see is the delivery of whatever rejuvenation technology through the stratum corneum, the outermost layer of the skin, which is very well designed to work as a barrier to protect the skin from potential harm or infections. Our peptide, as a reference, is considered small (10 amino acids) and we were fortunate to validate its ability to penetrate through the stratum corneum barrier and into the dermal layer. Larger molecules may face additional challenges in penetratration and to promote their effects in deeper skin layers.

Importantly, to be able to validate any technology to promote skin rejuvenation it is not a trivial process. Previous experience has shown that skin aging is a little bit different from aging in other tissues. Therefore, it will be important to validate other strategies while considering the important drivers for skin aging by testing on aged models that replicate chronological aging such as UV exposure, oxidative stress, and pollution, and not on less important drivers such as oncogene-induced and chemically-induced senescence. The ability to replicate these models required years of optimization and it is an ongoing process when you start considering not only the influence of age, but also how the diverse genetic background plays a significant role in this matter.

What is the future of OneSkin Technologies beyond your first senolytic product?

OneSkin's main goal is to build the first skincare line targeting cellular senescence and bring its own products to the market. We believe there is no proposal out there, focusing on skin, that tackles aging from its cause as we do. This makes us confident in the value our products will bring to consumers. After the first validation for aesthetic skin rejuvenation, we are going after other age-related skin disorders and eventually, age-related disorders beyond the skin. An oral application of our peptide is another avenue to be explored. As we intend to keep our focus mainly towards skin applications, we envision to explore these additional indications through partnerships with pharma or other longevity companies. Finally, OneSkin's main asset is our screening and validation platform, which will constantly screen and identify new leads, be them small molecules, peptides, natural compounds or combinations thereof, to target cellular senescence and senescence-associated diseases. We are determined to work to position our technologies in the forefront of the future therapies for aging and longevity.