A zinc complex was recently developed to help restore functional elastin in photodamaged and aging skin. Until this discovery, there was no method to regenerate damaged elastin and restore resiliency to photoaged skin. Studies in the murine model demonstrated the ability of this zinc complex to increase epidermal thickness, augment hypodermal fat and tropoelastin mRNA as well as increase elastin content. This article summarized the proceedings of an interactive dialogue among leading clinicians who reviewed these findings and made recommendations for future research.
Introducing the Panel of Experts
On Feb. 24, 2007, a Clinical Council on Skin and Aging convened in Miami, Florida, to discuss skin aging and the role of elastin in maintaining skin elasticity and new research findings with regard to elastin regeneration. Participants were thought leaders in clinical and cosmetic dermatology and plastic surgery as well as scientists who had participated in the development of a new zinc complex.
The purpose of this meeting was to initiate an interactive dialogue among clinicians and researchers to review the science of elastin regeneration and its role in photodamaged and aging skin. It was hoped that this “bench-to-clinic” discourse would lead to fruitful discussions of both key findings and limitations of the existing data as well as to suggestions for future research in the breakthrough therapeutic area of elastin regeneration.
Physician participants in the Clinical Council were: Leslie Baumann, M.D., Zoe Draelos, M.D., Patricia Farris, M.D., Timothy Flynn, M.D., Michael Kane, M.D., Wendy Lee, M.D., Victor Narurkar, M.D., Susan Taylor, M.D., Susan Weinkle, M.D., Jessica Wu, M.D., Mina Yaar, M.D., and Connie Ho, M.D.
Elastin is a connective tissue protein present in the extracellular matrix of tissues that undergo repeated physical deformations including skin, lung and vein tissue.1 Elastin is composed of enzymatically cross-linked tropoelastin, its soluble precursor.2 Research in vascular biology has focused on the significant clinical problem of loss of elastic tissue, and these discoveries applied to skin biology.
Skin ages in two overlapping ways, intrinsically (due to chronologic aging) and extrinsically (known as premature, environmentally-induced aging). While chronologic aging is universal, inevitable over time and occurs in all organ systems, extrinsic aging of the skin can largely be prevented by avoiding environmental damage from ultraviolet radiation, smoking and other forms of pollution.
An estimated 80% of visible facial aging is due to chronic UV exposure.3 Extrinsic aging, is superimposed upon the normal intrinsic aging process and leads to the wrinkles, loss of skin tone and dyspigmentation that are typically associated with age. In contrast, intrinsic aging causes relatively minor impacts on the skin’s appearance such as fine wrinkles, dryness and thinning as well as loss of the supporting subdermal fat pad.4 Clinicians see these differences every day while comparing the appearance of environmentally and sun-exposed skin to protected areas of the body.
Intrinsic and extrinsic aging produce differences in the dermal matrix. Collagen and elastin fibers, along with glycosaminoglycans, are the primary structural components of the dermis. In photoaged skin, the number of dermal fibroblasts decreases with a corresponding decrease in the synthesis of both collagen and elastin, leading to wrinkles and a loss of resilience.4 Fisher et al demonstrated that UV radiation also causes the degeneration of the dermal matrix via increased activity of matrix metalloproteinases (MMPs). Our approach to collagen loss has been the replacement of lost volume with collagen-based filling agents.5 As a result of the increased understanding of the pathogenesis of UV-induced collagen damage and loss, an additional approach to facial skin aging has been the use of various strategies designed to prevent the induction of collagenase and other MMPs. However, there have been no parallel advances in protecting or replacing lost or damaged elastin.
In the skin, elastin functions in conjunction with collagen to confer mechanical properties, notably tensile strength, elasticity and resilience. Elastin fibers are responsible for the skin’s ability to recoil after distension, a property essential to dynamic connective tissues including arteries.6 Elastin, as one of the participants noted, is not a fiber. Elastic fibers are assembled from tropoelastin, the gene product of the human elastin gene, a single copy gene on chromosome 7. After its synthesis by fibroblasts, tropoelastin begins cross-linking at the cell surface on a scaffold of fibrillin-containing microfibrils and rapidly becomes an insoluble elastin fiber.6,7
Functional elastin production peaks near birth and the early neonatal period and is nearly nonexistent by maturity in the majority of tissues.8 Figure 1 a through d (below) shows the natural history of elastin fibers from 5 days of age, when there is virtually no amorphous elastin but rather large microfibril bundles, to 48 years of age, when amorphous elastin and variable microfibrils with precipitates within the elastin fibers are apparent.
In normal skin with low sun exposure, the skin’s elastic network contains three types of fibers: fine oxytalan fibers in the papillary dermis, thicker elaunin fibers in the superficial reticular dermis and mature elastic fibers, interspersed among the collagen bundles in the deep dermis.9 In aging but protected skin, the elaunic and deep dermal elastic fibers are decreased. In one study, electron microscopy revealed the disappearance of oxytalan microfibrils, and progressive lysis of the amorphous matrix in the elaunin and elastic fibers along with cyst formation.9
In photoaging, one of the most important histopathologic markers is solar elastosis, a significant accumulation of amorphous elastotic material in the papillary dermis which differs in appearance to that of normal functional elastin.3,10 This abnormal elastogenesis may in fact be a reparative effort.11 Thus, unlike with collagen, photoaging leads not to a net loss of elastic fiber tissue but rather to a loss of functional elastin.
Scanning electron microscopy revealed increased complexity of the shape and arrangement of the elastic fibers in photodamaged skin, accompanied by a decrease in interfibrillar areas. Transmission electron microscopy revealed a decrease in microfibrils and increased complexity of the shape and arrangement of the elastic fibers, decreased amorphous elastic material and increased numbers of electron-dense inclusions and vesicular structures.12 As with collagen, MMPs also appear to play a role in elastin degradation.13
Elastin Synthesis and Replacement
Unlike with collagen, which can be replaced in the dermis using a variety of collagen fillers, to date there has been no way either to replace lost elastin with fillers or agents to stimulate its repair or to retard its degradation. It has been shown by several investigators that elastin regulation is mediated by several intra- and extra-cellular pathways (Figure 2, above), including epidermal growth factor receptors (EGFr) and insulin-like growth factor-I receptors (IGF-1-r) as well as insulin signaling and transforming growth factor beta-I (TGF-B1).14-16 The production of functional elastin, as opposed to an undesirable perturbed matrix, is dependent upon simultaneous inhibition and activation of the various pathways.
Zinc has many roles in biological membranes, cell receptors and proteins.17 The working hypothesis was based on zinc’s wide cellular and tissue effects in pathways that are also involved in elastin production:
1. It has been shown to affect EGFr-stimulated intracellular signaling which influences a variety of cellular functions including mitogenesis and apoptosis, protein secretion and differentiation or dedifferentiation.18
2. Zinc increases tyrosinase phosphorylation and activates intracellular signaling that includes MAP kinase activity, essential for co-signaling in extracellular matrix production.16,19
3. It possess insulin-like effects in lipogenesis, glucose transport and glucose oxidation in adipocytes20,21,22 and has been shown to potentiate the mitogenic signaling of insulin.23
4. It has been suggested that zinc may be involved in the insulin-signaling pathway which has a co-signaling role in extracellular matrix pathways.24
5. In bone tissue culture studies, the presence of zinc was shown to significantly increase IGF-1, TGF-B1 concentrations and protein.25 IGF-1 has been shown to increase elastin gene transcription14 while TGF-_ appears to be involved in elastin deposition during tissue repair and other conditions and to stabilize elastin mRNA.15 TGF-B1 also has effects on proteins of the SMAD family, which help regulate the genes for collagen.26
Zinc Transport Complex: Preclinical Findings
Zinc’s effects on all of these pathways, as well as previous in vitro vascular tissue studies in the setting of re-stenosis following stenting procedures, which demonstrated increased elastin production following treatment with a zinc complex,16 it was hypothesized that treatment with specific concentrations of ionic zinc could stabilize the EGFr, leading to epidermal thickening, regulation of insulin signaling pathways which lead to augmentation of hypodermal fat, and finally, production of functional elastin through these multiple pathways, both intracellular and extracellular.
Dr. Michael Dake noted that the desired effects on functional elastin production occurred within a tight therapeutic range and were obtained only with an optimal zinc concentration. Too high a concentration actually stimulated elastase production, while too low a concentration had no effect on tropoelastin mRNA.16
Randomized, controlled studies in mice showed that 21 days of treatment with 20 L 1.0 M Zn led to a 50% increase in epidermal thickness versus control and 151% increase in hypodermal fat (p<0.05). In the same model, cross-sectional analysis revealed a 53% increase in elastin with the Zn complex versus control (p=0.0001). (See Figure 3). Future studies will be required to ensure that the elastin that is produced is functional.
Inhibition of elastase was studied to assess the impact of zinc on elastase activity. It was demonstrated that zinc salts stabilize, then destabilize, and then restabilize elastase function according to the zinc concentration. Zinc (and other divalent cations including calcium and magnesium) was found to induce elastase activity at very low (<0.01 M) concentrations. Concentrations from 0.01 M to 0.75 M inhibited elastase while concentrations > 0.75 M again induced elastase activity. Elastase was found to be most consistently inhibited by zinc ions. 16
All of the effects, including increases in tropoelastin mRNA, epidermal thickness and fat augmentation and elastase demonstrated in the preclinical studies were found to be dose-dependent. Much participant discussion centered on the unique penetration technology that permits delivery of the zinc formulation into the skin. It was agreed that further elucidation of the delivery and activity of the zinc complex within the skin would be of real interest to the dermatologic community.
The first clinical trial was a prospective, randomized, double-blind, controlled study conducted by James Leyden, M.D. Subjects included 26 females aged 37 to 60 years (mean=50) who were regular users of eye creams (whose used had to be discontinued 7 days prior to study initiation). Users of retinoids and steroids were excluded. Subjects were randomized to apply the zinc complex twice daily to the periorbital area of one eye. The contralateral eye was treated with a commercially available copper compound that was considered to be the benchmark of cosmeceutical efficacy at the time of the study and had been shown to provide cosmetic benefits without any change in functional elasticity. Patients were provided a list of cleansers and toners that could be used, and no cosmetic procedures were permitted during the study.
Endpoints included changes from baseline and changes in comparison to benchmark control. Assessments were made by snap test and suction cup probe to measure skin elasticity as well as blinded dermatologist assessment of aesthetic improvement. Follow-up was at weeks 1, 2, and 4. Subjects were evaluated by a dermatologist at each time point by means of a 1 to 9 scale for fine and coarse wrinkling, undereye skin laxity, puffiness, crepe-like eyelid appearance, intensity of dark under eye circles, surface roughness/dryness and periorbital hollowness. Measurements were made under controlled room atmospheric conditions.
Snap time testing was used to demonstrate changes in functional elastin. In this test, the undereye skin is deformed by fixed distension and the amount of time required to return to its resting contour is recorded. A caveat noted by several Clinical Council members is that snap testing can also be an indicator of enhanced moisturization unrelated to elastin function. However, since each patient functioned as her own control, the control side provided a clinical benchmark for active moisturizing. The snap test was administered and videotaped so as to provide a frame-by-frame analysis performed by a blinded examiner. The Wilcoxon matched-pair signed rank test was used to examine significance at the 0.05 level. By week 4, a 44% improvement in snap time (2.1 seconds, p< 0.05)) was demonstrated with the zinc complex versus a 6% improvement (p>0.05 vs baseline) with the copper peptide.
Elasticity was also assessed by means of a DermaLab (CyberDerm) suction cup probe at baseline and weeks 1, 2, and 4. This instrument features a lightweight probe which is glued to the skin to eliminate movement artifacts. With the probe in place, negative pressure elevates the skin, and the differential negative pressure needed to lift the skin a predetermined distance is used to assess differences in skin elasticity. A statistically significant improvement (p<0.05) was demonstrated at weeks 2 and 4. The meeting participants discussed some of the drawbacks associated with the suction cup and inter-patient variability in results due to differences in anatomy and probe placement. It was generally agreed that the snap test results were more reproducible than suction cup data in this setting, because the suction cup testing method can also produce improved results based on skin moisturization rather than from an elastic tissue effect.
At 4 weeks, a blinded dermatologist assessment found superior aesthetic improvements with the zinc complex for fine lines, coarse wrinkles, crepey appearance, puffiness, skin laxity and dark circles. Figure 4, above, shows the relative improvements obtained for each of these parameters. In addition, improvements in eye contours and aesthetic appearance were also seen.
The findings regarding dark circle improvement could not be explained by the current understanding of the technology. A vehicle-controlled study of this phenomenon would be of interest since dark circles are a frequent and distressing appearance issue with no universally effective treatment at present.
Several participants noted that the increases in elastin appeared to be very rapid and wondered whether it continued to increase. Dr. Drake noted that maximum elastic tissue production occurred at 4 to 6 weeks before it reached a plateau, although the earliest histologic changes could be seen at approximately 2 weeks, which was similar to the time to first effects were seen in the animal studies. Mr. Browne said that an as-yet-unelucidated physiologic counter-mechanism led to this plateau in elastin, followed by increased elastase production.
Unfortunately, it is impossible to return to adolescent levels of functional elastin as patients might wish. However, he noted in response to a question, no matter how much of the formulation a consumer used in search of dramatic results, it would not be enough to exceed the optimal zinc concentration and lead to elastase production.
In reply to a query as to the timing of the increase in epidermal thickness that is obtainable with retinoids, one participant said that within 2 to 4 days, 100% of the epidermal cells that are capable of dividing enter the cell cycle. The flaking and scaling that are emblematic of topical retinoids are due to pressure from the dividing cells in lower levels of the skin. This participant expressed some surprise that epidermal thickening did not appear earlier than 4 weeks, although he did agree that differences between mouse skin and human skin might explain why epidermal thickening was not apparent before week 4.
In reply to questions regarding irritation, Mr. Browne noted that irritation was similar with both the zinc formulation and the copper peptide control. However, several participants suggested that it would be important to have a vehicle control
Another discussion involved the placement of the suction cup. It was suggested that subject anatomical differences as well as differences merely in hydration might compromise the reliability of the results. Dr. Drake agreed that the suction cup test did not appear to be as consistent from patient to patient as would be desirable.
There was also discussion regarding the subjects’ self-reports. Mr. Browne said that the patients had generally been pleased with the treatment and had been able to correctly identify the treated eye. However, many patients did not like the heavy emollient base that was used initially and so an off-the-shelf base was substituted. It was noted that patients, while pleased, unrealistically expected a “Botox-type response.”
Lastly, one council member questioned whether a power analysis had been performed to analyze the number of subjects who would be needed to achieve statistical significance. Dr. Drake said that it had been.
Changes in tropoelastin and elastin content were measured in a prospective, randomized, double-blind clinical study of 10 female subjects aged 50 to 64 years (mean=55). Patients applied the zinc complex twice daily for 2 weeks. Endpoints were changes in tropoelastin content using a tropoelastin antibody stain and changes in elastin content using Verhoeff-Van Gieson (VVG) stain. At 4 weeks, the tropoelastin content, measured as mg tropoelastin/mg of biopsied tissue, was shown to increase by 40% compared to baseline (p<0.001). Figure 5 shows the increased elastin fibers at 2 weeks compared to baseline.
Silicone Replica Analysis Study
In another prospective, randomized, double-blind clinical study, 27 female patients were evaluated for safety and effects of the two zinc complex formulations versus a control eye cream on the appearance of fine lines and wrinkles in the orbital area after 4 weeks of use. The primary efficacy endpoints were blinded dermatologist assessments of silicone skin replica images obtained from the “crow’s foot” area of each eye as well as aesthetic parameters. Patients discontinued using their usual eye cream for 48 hours prior to study initiation. Improvements in the density, number and depth of lateral canthal lines were observed after 2 weeks of use and continued throughout the study. Figure 6, below, shows the improvements in both gross undereye appearance and silicone replica contours after 4 weeks of product application. In addition, blinded dermatologist assessment revealed a 37% decrease in coarse wrinkles, a 29% decrease in fine lines, a 30% decrease in infraorbital puffiness and a 43% decrease in infraorbital laxity. All findings were significant versus baseline (p<0.01).
A question was raised as to whether subjects used the zinc complex on the day that the silicone skin replica analysis was carried out. If the skin was hydrated, particularly in a way that also lent some occlusion, there might be an apparent decrease in wrinkles visible by skin replica analysis that was in fact an artifact of the hydration or occlusion. The subjects had not used the zinc complex on the day that the silicone replica analysis was performed. It was also noted that larger pores or lesions, such as seborrheic keratoses or increased facial hair, might influence the findings from the skin replica analysis.
Another participant asked whether the fact that the product’s increased fat might not actually lead to increased fat deposition under the eye, leading to increased puffiness in this area. Dr. Drake noted that the increase in hypodermal fat was demonstrated in the murine model only and that in humans only epidermal thickening and increased elastin were demonstrable in an objective, quantitative way.
In the absence of an indisputably agreed-upon gold standard by which to measure elasticity, photographs are essential. It was generally agreed by the council members that excellent clinical photographs, in addition to high quality cellular and molecular studies, are often the most convincing evidence for dermatologists. However, to be meaningful, these photographs need to be taken under extremely well-controlled conditions. These include the use of a fixed camera, seat and head holder as well as a 10-megapixel image size. These studies are currently underway.
Dermatologists and their patients have long desired a product that could help restore lost cutaneous elasticity much as lost facial volume can be improved with the use of products designed to restore lost collagen.
These trials of a topical generally recognized as safe zinc formulation appeared to increase cutaneous levels of elastin in murine models. Assessments of aesthetic parameters also showed significant improvements at weeks of treatment. However, it must be emphasized the this field of research is still in its infancy and that research is ongoing based on the input of the participants in the clinical council and others in the fields of clinical and cosmetic dermatology.
All physicians who participated in the roundtable discussion expressed interest in this new technology and its ability meet an important and unfilled need in dermal matrix regeneration and made a number of suggestions for new research directions to augment and clarify the existing science.