While Cer are essential components of the epidermal permeability barrier, decreased Cer content and changes in Cer molecular distribution also have been demonstrated in several dermatoses, including atopic dermatitis [32
] and psoriasis [36
]. Not surprisingly, both atopic dermatitis and psoriasis display phenotype-dependent abnormalities in barrier function [37
]. Accordingly, in vivo
studies in both experimental models and preliminary human studies have demonstrated that topical Cer-containing lipid mixtures can improve barrier function [12
]. More importantly, Cer-containing lipid mixtures currently are being assessed for their potential use as primary and/or ancillary therapy of atopic dermatitis. Because of the potential risks and high cost of natural- and skin-identical synthetic Cer, respectively, chemical synthetic mimics, or pseudo-Cer, have been developed as Cer-alternatives. Whereas natural Cer have potentially deleterious effects, including inhibition of cellular proliferation and induction of apoptosis, in a variety of cells [6
], including keratinocytes [21
], certain metabolic and catabolic pathways can suppress potential Cer accumulation, thereby protecting cells from Cer-induced apoptosis [20
]. In addition, natural Cer are nearly cell-impermeant suggesting that the quantity of natural Cer absorbed into the nucleated cell layers may not be sufficient to induce apoptosis.
Despite the apparent efficacy of pseudo-Cer in the treatment of atopic dermatitis [4
], whether or not pseudo-Cer(s) exhibit(s) similar effects as seen with natural Cer on cellular growth and apoptosis has not been reported. Our present studies demonstrate that two pseudo-Cer, BIO391 and PC-104, have fewer adverse effects on cell growth and toxicity than exhibited by either comparable concentrations of nature-identical or cell-permeant Cer in intact CHK. Likewise, the two pseudo-Cer exhibited a lesser propensity to induce apoptosis-related, membrane depolarization in membrane-permeabilized, semi-intact cells. However, these studies do not exclude possible adverse effects of other, chemically-unrelated pseudo-Cer that were not tested here. Lack of toxicity to CHK at these concentrations, though relevant for topical therapy in human subjects [10
], does not, however, exclude the possibility of toxicity by these pseudo-Cer to other cell types should substantial absorption occur. Since even more pseudo-Cer could penetrate into skin with barrier defects, we assessed unimpeded access of these agents to undifferentiated cultured keratinocytes, which represent the cells of nucleated cell layers in epidermis and do not have permeability barrier structures which could exclude pseudo-Cer from the nucleated layers of the epidermis. Our results demonstrate that pseudo-Cer do not show significant toxicity at concentrations that assume approximately 100% absorption of the agent applied topically. In fact, the doses (up to 25 μM) tested in this study represent a similar concentration range of pseudo-Cer agent used for topical therapy (i.e
., 1–2%). Thus, even if 100% of topical applied pseudo-Cer penetrate into skin, it is not likely to show significant cell toxicities as demonstrated in this study. Moreover, PC-104 is not a topical irritant or sensitizer after applications at high concentrations, under occlusion, to intact and wounded rodent skin; or when instilled into rabbit eyes, and it is not mutagenic in the Ames (S. typhimurium) assay (FDA 510K Notification for EpiCeram Skin Barrier Emulsion, April 12, 2006). In addition, PC-104 has been widely used for over five years in the U.S., without reports of toxicity, in a triple-lipid formulation (TriCeram, Osmotics Corp.). Finally, it should be noted that TriCeram has been extensively employed as ancillary therapy for atopic dermatitis [4
], a disease characterized by defective barrier function (op cit
), without reports of cutaneous or extracutaneous side effects.
Even more extensive in vivo preclinical safety studies were performed on BIO391, which likewise revealed no evidence of toxicity, including oral administration (LD50 > 2000 mg/kg); lack of acute toxicity to Daphnis magna: neither skin nor eye irritation; and no evidence of skin sensitization; and no evidence of mutagenicity (tested according to OECD guidelines #423, 402, 404, 405, 429, and 471, April, 2006). Thus, neither cutaneous nor extracutaneous toxicity appears likely to occur with either of these compounds.
We did not address whether these and other pseudo-Cer are metabolized into potentially-toxic metabolites. However, as both of these pseudo-Cer are amphiphilic, highly hydrophobic (log P o/w > 13) compounds with molecular weight > 500 [41
], substantial penetration into deeper epidermal layers, a prerequisite for consequent metabolic conversion and/or systemic uptake, appears unlikely. Moreover, BIO391 reportedly is very stable over a pH range of 5–7 [14
], excluding the possibility of a non-enzymatic, pH-driven degradation under physiologic or pathologic conditions.
Finally, prior studies demonstrated that Cer containing 2-OH fatty acid (2-OH Cer) are less potent than Cer containing non-hydroxy fatty acid for inducing apoptosis in U937 cells [42
]. However, since both BIO391 and PC-104 are designed as Cer-containing, non-OH fatty acid mimetics, we did not compare the effects of 2-OH Cer in this study. Although it has not been determined whether 2-OH Cer either have less potent toxicities in CHK or improve epidermal permeability barrier function, 2-OH Cer mimetics pseudo-Cer may further assure safety for these therapeutic agents.
In summary, two chemically-unrelated, synthetic Cer mimics or pseudo-Cer, N-palmitoyl-4-hydroxy-L-proline palmitoyl ester (BIO391) and 1,3-bis(N-2-(hydroxyethyl)palmitoylamino)-2-hydroxypropane (PC-104), display lesser effects on CHK growth and viability than either exogenous natural or cell-permeant Cer, providing further evidence for the safety of these two agents when incorporated into topical therapeutics. Yet, further assessments of the safety pseudo-Cer still need to be performed in vivo.