Oiligonucelotides And Skin

The skin is the largest organ of the human body, covering nearly 2m2 of surface area on an average adult and receiving about one-third of all blood circulating through the body. The primary function of the skin is to act as the body's major barrier. It maintains the fluid homeostasis within the body while preventing compounds from entering from the external environment. To do this, the skin has developed two layers, the epidermis and the dermis. The epidermis can be subdivided into the viable epidermis and the stratum corneum (5).

The outermost layer of the skin, the stratum cornuem, is approx 15-20 ^m thick in human skin and consists of terminally differentiated keratinocytes

From: Methods in Molecular Medicine, Vol. 106: Antisense Therapeutics, Second Edition Edited by: I. Phillips © Humana Press Inc., Totowa, NJ

(corneocytes) that are embedded in a matrix of lipid bilayers (6). It is the skin's principle barrier and can be thought of as a brick wall, with corneocytes serving as the bricks and lipids surrounding them as mortar (7). The viable epidermis is located just below the stratum corneum, and its principal function is the production of stratum corneum (8). The dermis is the innermost layer, consisting mainly of collagen fibers in an aqueous gel matrix that imparts elastic properties to the skin. It contains blood vessels, lymphatics, and nerve endings and is the physiological support mechanism for the epidermis. The skin also contains hair follicles and sebaceous glands that can act as shunts for transdermal penetration of chemicals. The surface area of these appendages, however, is quite small when compared with the total surface area of skin, and, therefore, they have a minor role in penetration (9).

Keratinocytes are formed at the epidermal/dermal junction. As they begin the process of terminal differentiation, they are replaced with newer keratinocytes. As differentiation occurs, cells move away from the epidermal/ dermal barrier toward the stratum corneum. By the time that they have reached the stratum corneum, they have elongated and flattened, lost their nuclei and other organelles, and are surrounded by a thick band of protein forming a corni-fied envelope. After approx 2 wk in the stratum corneum, the corneocytes reach the outside of the stratum corneum and are sloughed off in a process called desquamation (10).

The lipids associated with keratinocytes change as the cells differentiate. Unlike most biological tissues, the stratum corneum contains no phospholipids but, instead, ceramines, cholesterol, fatty acids, sterol esters, and cholesteryl sulfate (8,11). These lipids form the bilayer that becomes the major barrier to water and water-soluble chemicals. For appreciable quantities of chemicals to cross the skin, therefore, a permeant must pass through the stratum corneum's brick wall. Evidence has shown that for a molecule to transverse this barrier successfully, it must rely on partitioning into the lipid mortar surrounding the keratinocytes. It then remains in the lipids and diffuses through the thickness of the stratum corneum. On successfully crossing the stratum corneum, the permeant must then diffuse into the more hydrophilic viable epidermis before proceeding to the blood vessels in the dermis. The need to partition into stratum corneum lipids and then into the hydrophilic bloodstream favors molecules that are moderately lipophilic (12).

Movement through the skin is different from many other tissues in that it relies on passive diffusion instead of active transport. Flux across the skin can be described by Fick's law, which demonstrates a linear relationship among penetration, donor concentration, and exposed area. Furthermore, smaller and uncharged chemicals tend to penetrate better than comparable larger and charged molecules. Several additional factors affect the feasibility of the therapeutic delivery of drugs through the skin, including the surface area of the delivery patch and dose of drug needed. A transdermal patch cannot be too large; generally, it is assumed that 50 cm2 should be the largest coverage area. Based on Fick's law, chemical flux is linearly related to surface area. Therefore, calculations have shown that the maximum dose feasibly delivered for chemicals that readily penetrate the skin should be in the low milligram range (13).

Given the information provided above, it seems obvious that delivery of antisense oligonucleotides (AS-ODNs) through the skin is unlikely to succeed. They are large, highly charged, and hydrophilic and require doses in humans that are too large to be feasible by transdermal delivery. Thus, sufficient quantities of oligonucleotides would not be able to penetrate through the stratum corneum. Two approaches have been used to reduce these problems and make transdermal delivery more feasible. The first is to improve delivery across the skin using chemical and physical penetration enhancers, and the second is to alter the oligonucleotide chemistry to increase potency, thereby reducing the quantity that must be delivered. The latter is not discussed in this chapter but is addressed in other areas of this book.

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