Chronic insults to the skin such as ultraviolet light, ozone, cigarette smoke, pollutants and other natural and synthetic environmental stimuli lead to cumulative damage, and can result in photoaging and "heliodermatitis".¹ Chronic ultraviolet sun exposure leads to clinical changes in the skin such as laxity, roughness, dryness, sallowness, pigmentation, telangectasia and wrinkles.² Reactive oxygen species such as free radicals unquestionably produce oxidative damage in skin. Ultraviolet light contributes directly to photodamage, not only by generation of reactive oxygen species but also by depression of antioxidant levels.³ Antioxidants are obviously necessary for neutralizing oxygen molecular species such as oxygen free radicals, which damage and destroy skin. Vitamin C (ascorbic acid) has been shown to have antioxidant effects as well as a role in collagen stimulation. Ascorbic acid appears to influence production of collagen by post-translational and transcriptional mechanisms.⁴ This is thought to occur by ascorbate stimulating collagen synthesis directly and specifically activating collagen gene regulation, both by increasing transcription rate and by stabilizing pro-collagen mRNA, therefore, genetically signaling collagen synthesis.^{5 6 7 8 9} Another mechanism is initiation of lipid peroxidation, which leads to an increase in a byproduct, malondialdehyde, which somehow stimulates collagen gene expression.¹⁰

Ultraviolet light exposure depletes up to two-thirds of cutaneous Vitamin C stores.⁴ Cutaneous levels not obtainable by ingestion of Vitamin C can be reached with topical application. Topical Vitamin C provides more than twenty times the amount of Vitamin C found in normal skin.^{16 17} Ascorbic acid stereoisomers D-ascorbic acid and L-ascorbic acid exist, but only the L-ascorbic acid form can be used by the body.^{16 18} This stable form of L-ascorbate has allowed pharmacological levels of Vitamin C penetration targeted directly into the skin by topical application to effect antioxidant and collagen stimulation. Because topical Vitamin C does not absorb light in UVB/UVA range, it is not a sunscreen but exerts its effects by neutralizing oxygen free radicals.^{20 21}

Skin inflammation, which is mediated by reactive oxygen species, has been reported to be alleviated by topical use of Vitamin C on ultraviolet radiation-induced erythema on porcine and human skin.¹⁴ This same study also showed a protective effect on the inflammatory response when applied, even after sun exposure from ultraviolet sunburn.¹⁴ Topical Vitamin C has also been used as a priming agent as well as a postoperative agent in laser resurfacing erythema treatment.³⁰ Furthermore, the introduction of a stable preparation of L-ascorbic acid is now available that can penetrate the skin, delivering L-ascorbate to the epidermis and dermis. It is anticipated that this enhanced delivery technology utilizing a unique formulation of L-ascorbic acid, zinc sulfate and L-tyrosine, will show an enhanced and longer-lasting effect on photodamage, prevention and aging. The central hypothesis of this study is that a daily regimen of topical Vitamin C will lead to subjective as well as objective improvement in human facial photoaging/photodamage. Optical profilometry is an objective method for quantification of facial wrinkles.³¹ Skin replica analysis of photodamaged skin was well described and utilized in previous studies with the use oftretinoin.^{32 33} No previous studies have utilized this technology with the use of topical Vitamin C.

Main Outcome Measures: Specific clinical parameters evaluated: fine wrinkling, tactile roughness, visual dryness, coarse rhytids, telangectasia, laxity/tone, pigmentation, keratoses and sallowness. Each of these parameters were graded on a 0 to 9 point scale (0=None, 1-3=Mild, 4-6=Moderate and 7-9=Severe). Reference photographs were used to standardize grading criteria. Overall investigator global scores were compared to baseline as: excellent (much improved), good (improved), fair (slightly improved), no change or worse. Patient self-appraisal questionnaires rated the degree of improvement (much improved, improved, slightly improved, no change or worse) and reported side effects (burning, stinging, redness, peeling, dryness, discoloration, itching, rash). Standard photography, including AP as well as left and right oblique views, were obtained at baseline to facilitate subsequent clinical evaluations and at the end of therapy for comparison. Optical profilometry analysis of skin surface replicas of the crow’s feet region were obtained, comparing baseline to end-of-study specimens. Utilizing the Magiscan System, the resulting image was digitally analyzed, and numeric values were assigned to reflect surface features. The parameters obtained included: Rz, Ra, and Shadows. These values provided objective data which documents pre-and post-treatment texture changes.

Optical profilometry analysis of skin surface replicas of the crow’s feet region were obtained, comparing baseline to end-of-study specimens. Silicone skin surface replicas were taken from the periorbital crow’s feet region at identical sites bilaterally by the same technician. Alcohol skin cleansing of the periorbital region was utilized before application of the adhesive rings and silicone impression material (CuDerm Corporation, Dallas, TX). Precise application of the adhesive replica locating rings was aided with caliper measurements to insure consistent distances from reference points of the lateral orbital canthus and superior auricular tragus. This, as well as use of reference close-up Polaroid photographs with adhesive rings properly in place for each subject, facilitated relocating these sites for subsequent end-of-study samples. Results:

Three patients were eliminated from the study secondary to the inability to follow-up at designated study protocol periods. Seven additional patients were excluded from analysis because of breach in study protocol for active and control designations. Of the nineteen evaluable subjects, three were male (mean age=43) and sixteen were female (mean age=48). Ages ranged from 36 to 72 years. Pre-study data revealed: 63% of patients had a previous history of smoking, 52% utilized sunscreens on a regular basis, 52% admitted to excessive lifetime sun exposure. Side effects were mild and usually resolved within the first two months of therapy. Side effects included, in decreasing order of frequency: stinging (55%), erythema (24%), and dryness (.05%). All side effects were easily treated with moisturization. In no case was topical required or topical study regimen altered. The majority of initial improvement seen during this time period involved tactile roughness/texture and skin hydration changes.

Optical profilometry image analysis demonstrated a statistically significant 73.7% improvement in Rz and Shadows North-South values with active greater than control, as well as a trend for improvement in Rz North-South parameter showing a 68.4% active greater than control improvement. Clinical investigator assessment demonstrated statistically significant improvement with active greater than control for fine wrinkling (p=.002), tactile roughness (p=.035), coarse rhytids (p=.010), skin laxity/tone (p=.032), sallowness/yellowing (p=.031), and overall (p=.002). Patient questionnaire results demonstrated statistically significant improvement overall with 84.2% active greater than control (p=.002). Photographic assessment demonstrated statistically significant improvement with 57.9% active greater than control (p=.011).

In this study, a 3 month daily regimen of topical Vitamin C was shown to provide significant objective and subjective improvement of some of the changes associated with photodamaged facial skin. These changes were gradual and became progressively more evident as treatment continued. Clinical

assessment and patient questionnaire/self-appraisal have been the traditional areas of evaluation. These methods demonstrated significant differences from vehicle in fine wrinkling, tactile roughness, coarse rhytids, skin laxity/tone, allowness/yellowing, and overall skin improvement.

Clinical and patient self-appraisal showed an 84% correlation to predicting active versus control. Stinging did not have any significant correlation with active or control agents. The vehicle control agent (Cellex-C International, Toronto, Canada) was matched for colour, consistency, and pH to the active agent to ensure the blindness of the study. Stinging discrepancies were probably associated with variations in skin type and surface flora among subjects, as well as environmental exposure, cosmetic use, local tissue reactivity, variations in serum application, and moisturizer use.

Photographic assessment did show significant improvement with active greater than control but was found to have the least reliability (58%) in predicting active and control sides. Inherent limitations in photography include fluctuations in lighting, head position, facial expression and asymmetries. Consequently, the use of skin replica optical profilometry to complement subjective evaluations with an objective method for the quantification of skin surface texture changes with minimal variability or potential for bias was utilized.

Pre-and post-treatment comparison revealed significant improvement with active greater than control for Rz North-South (.032) and Shadows North-South (.032). In the case of Rz North-South, the values approached significance (p=.084). Therefore, overall active topical Vitamin C treated skin topography is smoother and less wrinkled than vehicle-control. The fact that Rz and Shadows represent fine to intermediate depth lines and that Rz is largely a measure of deep wrinkles, suggests that topical Vitamin C therapy had a more dramatic effect on superficial topography and less of an effect with major furrows and creases.

It should also be mentioned that there are an increased number of Vitamin C based topical cosmetics available on the market. It appears that not all preparations of topical Vitamin C are effective. Many of these products utilize derivatives, esters and analogs of Vitamin C that are either unable to penetrate into the skin, unable to be chemically converted to L-ascorbic acid (the only form that can be utilized by the body), and/or unable to be delivered in adequate concentration to have an effect. These ascorbic acid substitutes include ascorbyl palmitate, magnesium ascorbyl phosphate, ascorbic acid magnesium phosphate, which are easily stabilized but must be converted to L-ascorbate to be effectively useful. There is no direct evidence that ascorbic acid derivatives enter the skin in appreciable amounts, and it appears that their conversion to L-ascorbate is largely inefficient, therefore, precluding effective concentration delivery. "This three-month study evaluated and did show topographic improvement in facial photodamaged skin utilizing Cellex-C (Cellex-C International, Toronto, Canada)."