Clinical Evidence of Dermal and Epidermal Restructuring from a Biologically Active Growth Factor Serum for Skin Rejuvenation

posed of a network of collagen and elastin fibers. To maintain the integrity of these fibers, the ECM must also be maintained in a balanced state to ensure proper hydration. If the homeostatic state of the ECM becomes altered by intrinsic or extrinsic factors, the integrity of collagen and elastin becomes compromised, leading to the onset of various signs and symptoms of skin aging.Skin in a healthy state is characterized as being firm, smooth, strong, hydrated, and free of disease. To achieve all of these clinical endpoints requires comprehensive treatment options that are capable of simultaneously initiating multiple restorative biological pathways. The aim of this research was to assess the ability of a multi-modal topical treatment enabled by a combinatorial system of plant-derived and enzymatically derived stem cell technologies to restore skin to optimal health.

The Growth Factor test product’s multi-modal technologies included the following:

Growth Factor 1: A plant-derived fermented red ginseng extract rich in ginsenosides. Ginsenosides have been shown in bioassay testing to induce transforming growth factor beta-1 (TGF-β1) expression and upregulation of Type I collagen production in normal dermal fibroblasts to provide for anti-aging skin effects.⁸

Growth Factor 2: Disodium acetyl glucosamine phosphate is an enzymatically derived form of N-acetyl glucosamine that, when tested on human skin, has shown the ability to boost GAG production through enhancement of CD44 expression in the basal layer.⁹ In the same series of experiments, in-vitro testing also demonstrated the ability of this technology to increase fibroblast proliferation.⁹

Proprietary (ZPRO™) sericin is a biomimetic protein. Sericin has demonstrated the ability to upregulate the production of collagen and hyaluronic acid.^10-12

Dipeptide diaminobutyroyl benzylamide diacetate is a neuropeptide that acts as an antagonist of the muscular nicotinic acetylcholine receptor (nAChR), which prevents binding of acetylcholine to the receptor, so it remains closed. In a closed state, the uptake of sodium ions (Na+) is impeded and muscles stay relaxed. In human clinical testing, this technology demonstrated substantive benefits in reducing forehead wrinkles and crow’s feet.¹³

Angelica polymorpha sinensis root extract, commonly known as dong quai, is a technology that has been shown in in-vitro models to prevent cells from entering terminal senescence, stimulate ECM deposition, and inhibit the proposed

inflammatory enzyme cyclooxygenase-2 (COX-2).^14,15 In a cosmeceutical preparation tested on diabetic skin, it was proven to improve skin structure, directly translating into clinical improvement of elasticity, dryness, and appearance, as well as wound healing.14

Buddleja davidii meristem cell culture is a plant stem cell inherently rich in polyphenol content, specifically verbascoside, that mitigates inflammatory response and oxidative stress. Verbascoside has been shown to have a dose-dependent ability to decrease interleukin-8 (IL-8) expression on primary cultures of human keratinocytes.16 In a human pilot clinical trial, verbascoside’s antioxidant capacity demonstrated significant protection of skin lipids from oxidative stress, and a significant increase of the antioxidant resistance of the skin.16

Beta glucan is a natural polysaccharide commonly found in yeast, fungi, bacteria, and plants. An alternative to conventional beta glucan is a highly purified variant β-1,6- branched-β-1,3-glucan extracted from a unique fungal strain. The branched structure has shown higher immune-enhancing activity compared with other conventional β-1,3-glucan. Branched beta glucan has been shown in in-vitro models to achieve proliferation of human fibroblast cells, along with collagen synthesis.17 Testing on human skin showed superior skin hydration effects in comparison to hyaluronic acid.17

Study Design An Institutional Review Board (IRB)-approved, independent, open-label clinical study empaneled 44 subjects, of which 41 subjects completed the study. Those who completed the study ranged in age between 41 and 70 years, with a mean of 65.29 years. The proportions of patients who had skin types II, III, and IV were 41.5%, 48.8%, and 9.8%, respectively. All subjects were predetermined by expert grading as exhibiting moderate to severe fine lines and coarse wrinkles in the crow’s feet, and moderate to severe skin laxity/lack of firmness in the facial region. Subject questionnaires revealed that most subjects classified themselves as having at least moderate severity concerns regarding skin sagging, dryness, dullness, and roughness, as well as crow’s feet, fine lines, laugh lines, and deep wrinkles.Patients were instructed to apply a new topical formulation (ZO Growth Factor Serum) primarily composed of growth factors derived from plant and enzymatic sources twice daily for 12 weeks. Trained clinical researchers measured results through standardized objective clinical measures. A range of bioinstrumental measures (including a corneometer, a cutometer, high-resolution ultrasound, and 2D and 3D imaging analyses) were also employed. Additionally, subjective assess

ments were compiled through an administered questionnaire.The study protocol was approved by an independent IRB to ensure the protection of the rights, safety, and well-being of subjects. Prior to study initiation, the IRB reviewed and approved the study protocol and subsequent amendments, as well as the methods and materials used in obtaining and documenting informed consent of the subjects. The study was conducted in accordance with U.S. Food and Drug Administration (FDA) Good Clinical Practice and International Conference on Harmonization guidelines in as much as they apply to cosmetic research. Test Materials and Regimens After the initial screening visit, participants who consented to the study protocol went through a one-week washout period, during which they replaced their usual cleanser and sunscreen with investigator-supplied study support products: a gentle proprietary cleanser (ZO Skin Health Gentle Cleanser) and a sunscreen (ZO Skin Health Oclipse Sunscreen + Primer SPF 30). Investigators instructed participants to solely use the following daily skin treatment regimen during the 12-week treatment period:

Clinical Efficacy Assessments Visual analog scales (VAS) are used in clinical research to measure intensity or frequency of various symptoms, subjective characteristics, or attitudes that cannot be directly measured. VAS are reliable scales that are more sensitive to small changes than simple ordinal scales. When responding to VAS items, expert graders specify their level of agreement to a statement by indicating a position along a line (10 cm) between two endpoints or anchor responses. A VAS in which the ends of a 10-cm horizontal line were defined as extreme limits orientated from the left (best) to the right (worst) was used to evaluate efficacy parameters.At each subject’s baseline visit, and at follow-up visits 2, 4, 8, and 12 weeks thereafter, investigators evaluated each of the following parameters by employing VAS methodology:

At all study visits, researchers also used the following objective measuring tools:

A corneometer for hydration

A cutometer for firmness and elasticity

Ultrasound imaging (DermaScan, Cortex Technology, Hadsund, Denmark) for skin thickness/density

2D clinical photos of left, right, and center views (Clarity machine vision software from JADAK, Syracuse, NY)

A subset of 15 patients underwent 3D image/photo analysis (Antera 3D, Miravex Ltd., Dublin, Ireland) of the crow’s feet.

Additionally, all patients answered a detailed questionnaire about their perceptions of the treatment benefits at all follow-up visits. Statistical Analysis Methods Researchers used descriptive statistics to analyze subject demographics and subjective questionnaires. For expert clinical grading, instrumentation, and Antera image analysis, they used the paired T-test (monadic). Statistical significance was set at P less than 0.05.

Investigator All investigator-graded parameters showed statistically significant improvement over baseline at all follow-up visits. Week 12 results were as follows (Table 1): Instrumental All corneometer and cutometer measurements showed statistically significant improvement over baseline at all follow-up visits. Regarding corneometry at week 12, subjects had achieved a 40.51% mean improvement, with 85.4% of subjects showing improvement. The corresponding figures for cutometer scores were 10.09%/68.3%, respectively, for firmness, and 21.31%/65.9% for elasticity. Ultrasound measurements revealed a 33.39% mean increase in skin thickness versus baseline (P<0.001), with 93.8% of subjects showing improvement.