Topical delivery of retinyl ascorbate co-drug. 5. In vitro degradation studies.

Chemical and enzymatic hydrolysis of the co-drug of retinoic acid (vitamin A) and ascorbic acid (vitamin C) - retinyl ascorbate (RA-AsA)--have been studied. Firstly, the amount of protein and ester hydrolysis activity was determined in crude cellular extracts from freshly excised porcine ear skin (<3 h) and stored porcine ear skin (frozen >6 months) using ethyl butyrate as model substrate. The stability of RA-AsA was then determined in the crude cell extracts with and without additional antioxidants. Lastly, the enzymatic hydrolysis of RA-AsA and retinyl-2-carboxy-2-hydroxy-ethanoate were determined by incubating with porcine liver esterase - retinol palmitate and ascorbyl palmitate were included for comparison. Freshly excised skin contained higher amounts of active proteins than previously frozen skin. RA-AsA underwent hydrolytic reduction causing the AsA moiety to disintegrate due to the presence of free radicals in the media. An intermediate was produced that seemed to be cleaved by enzymes. Addition of ascorbic acid, as antioxidant, to the media of crude protein extracts decelerated the hydrolysis rate. This was supported when RA-AsA and retinyl-2-carboxy-2-hydroxy-ethanoate were incubated separately with pure esterase. There was approximately 5-fold more soluble protein per ml of cytosol in the fresh skin compared to the stored skin. Therefore, the amount of protein present within approximately 1.5 cm(2) of skin (average diffusion area in the Franz cells used in our skin penetration studies) was 0.06 mg cm(-2) and 0.01 mg cm(-2) for fresh and stored extracts, respectively.

Topical delivery of retinyl ascorbate co-drug: 6. Determination of toxic dose and antioxidant activity in cultured human epidermal keratinocytes.

The maximum effective dose of retinyl ascorbate and its potential therapeutic benefits against induced oxidative damage were assessed in vitro using cultured human epidermal keratinocytes. RA-AsA exhibited toxic effects at concentrations >6 microM. The findings indicate to the potency of RA-AsA as free radical scavenger and cell proliferation regulator.

Topical delivery of retinyl ascorbate co-drug. 2. Comparative skin tissue and keratin binding studies.

Retinyl ascorbate (RA-AsA), an ester co-drug of vitamins A (RA) and C (AsA), is proposed as a topical antioxidant/cell division regulator for reducing UV-induced generation of free radicals and disrupted dermal cell growth. The efficacy of dermatological agents is influenced by their retention within the skin, which is increased by the interaction with skin components. Keratin is the major protein (approximately 95%) in the skin, and this paper reports the binding of RA-AsA, RA, AsA, retinol, ascorbic acid palmitate and retinol palmitate to three tissues-human callus, pig ear skin and bovine horn keratin. Tissue samples were incubated with solutions of compounds and the uptake measured as the ratio of bound/free compound at equilibrium. Binding to keratin was assessed using delipidised tissue, and was much higher for the polar compounds, suggesting dipolar/H-bonding interaction. Binding strength was ranked as human > porcine > bovine, but there was no distinction for highly lipophilic compounds. The binding characteristic of native tissues was complicated by lipid content of the tissues. There seemed to be a dual effect. The binding of very lipophilic materials increased with lipid content, implying that a substantial amount is dissolved in the lipid matrix. For highly polar AsA, lipid content decreased the binding, suggesting that the lipid reduced the strong polar interactions with skin protein/keratin.