Therapeutic effect of NEO400, perillyl alcohol conjugated to linoleic acid, in a mouse model of UV-induced skin damage

Abstract

Excessive exposure to ultraviolet radiation (UVR) causes harmful effects on human skin. Pre-exposure application of sunscreen can be protective, but not after damage already has occurred. There is a need for agents that can be applied post-UVR exposure to repair the damage. We investigated a novel compound, NEO400, that appears to meet this medicinal need. NEO400 was created by conjugating linoleic acid to perillyl alcohol. UVR was repeatedly administered to the skin of mice over several weeks, where it caused the typical signs of UV damage, including scaling of the skin, DNA damage, and elevated levels of inflammatory cytokines. However, when NEO400 was applied immediately post-UVR, it triggered the appearance of markers for dermal stem cell proliferation, and no signs of skin damage emerged. Furthermore, when NEO400 was applied to skin that already had incurred significant damage, it accelerated skin healing. When applied individually, linoleic acid and perillyl alcohol were ineffective, indicating that they had to be conjugated in order to exert therapeutic efficacy. None of these skin-protective effects could be achieved with Aloe vera gel, a popular and widely used post-exposure remedy. Our study suggests that NEO400 holds potential as a regenerative treatment for excessively UVR-exposed skin.

Keywords: Aloe vera; UV radiation; linoleic acid; perillyl alcohol; skin damage.

FIGURE 1

Chemical structure of NEO400. Perillyl alcohol was covalently conjugated to linoleic acid to create NEO400 (Synthesis, validation, and quality control of NEO400 was performed by Norac Pharma).

FIGURE 2

Outline of treatment schedules. In all cases, mice received 3× weekly treatments, similar to schedules previously established by other groups., For Schedule A, treatment was performed for a total of five consecutive weeks. Exposure to UV light (of the back of the animal) was for 10 min in each session and was followed either by no additional treatment (or vehicle only) or by topical application of POH, LA, Aloe vera, sunblock, or NEO400. At the end of this 5‐week regimen, mice were evaluated and biological responses investigated. For Schedule B, mice received the same 3× per week UVR treatment as in Schedule A, but without any topical applications during the first five consecutive weeks. Thereafter, another 4 weeks of 3× per week UVR was added, but this time each UVR exposure was followed by topical treatment with NEO400, Aloe vera, or vehicle only. In both schedules, each individual treatment was applied to three mice (n = 3) in parallel.

FIGURE 3

Macroscopic appearance of UVR‐exposed skin. (A) A total of 18 mice were separated into six treatment groups, where each UVR exposure was immediately followed by topical treatment over the course of 5 weeks (as per Schedule A). For each group of three mice each, only one representative animal is shown before the onset of any treatments. After completion of the 5‐week treatment, photos were again taken, and photos of all three mice in each group are shown. (B) The extent of skin damage in each mouse shown in part A was evaluated by applying a skin damage score from 0 to 4, where 0 indicated no damage and 4 indicated very severe damage (see Materials and Methods). Shown are the averages from each group of three mice. Except for NEO400, none of the other treatments achieved any significant reduction in the skin damage score as compared to UV + Vehicle, and skin damage in all of them was significantly worse as compared to the UV + NEO400 group. ***p < 0.001. (C) Three mice per treatment group received the 5‐week exposure to UVR without any post‐UVR treatment, which resulted in pronounced skin damage (exemplary photo “N/A") at 5 weeks. Thereafter, the mice were subjected to continued 3× per week UVR, but this time each exposure was immediately followed by topical application of NEO400 or Aloe Vera (as per Schedule B). Two and four weeks later, photos were taken, and representative outcomes are shown, along with enlargements of the irradiated skin area.

FIGURE 4

Microscopic appearance of UVR‐exposed skin. Skin sections were prepared from mice subjected to the 5‐week treatment cycle of UVR‐only, or UVR + NEO400 (Schedule A). Histological characteristics were examined under the microscope. As exemplified in these images, the epidermis of UVR‐only skin showed signs of: (1) hypergranulosis, (2) dyskeratosis, (3) hyperplasic epidermis, and (4) abnormal structures with ductal differentiation, which were not apparent in skin sections from mice that had received NEO400 immediately following UVR exposure.

FIGURE 5

Cytokine levels after UVR exposure. Blood from mice subjected to the 5‐week treatment cycle of UVR with or without immediately following topical treatments (Schedule A) was analyzed for the levels of IL‐1β, IL‐6, and TNF‐α. Shown are averages from each group of three mice. *p < 0.05; **p < 0.01; ***p < 0.001; n.s.: No statistical difference (p > 0.05).

FIGURE 6

Markers of DNA damage after UVR exposure. DNA was extracted from the skin of mice subjected to the 5‐week treatment cycle of UVR, with or without immediately following topical treatments (Schedule A), and DNA content of 6‐4 PP and CPD was determined. Shown are averages from each group of three mice. *p < 0.05; ***p < 0.001; n.s., no statistical difference (p > 0.05).

FIGURE 7

Detection of skin stem cell markers. Skin sections were prepared from mice subjected to the 5‐week treatment cycle of UVR, with or without immediately following topical treatments (Schedule A), and subjected to staining with antibodies to follistatin or integrin α6. Size bar: 100 μM. Enlargements for each image are shown as well.