Although pharmacokinetic analysis of topical preparations may require the description of a relatively large number of compartments, this discussion is confined to the three outlined in Figure 215-1: (1) the skin surface, (2) the stratum corneum, and (3) the viable tissue. The formulation itself forms a reservoir, from which the compound must be released; in order to undergo percutaneous absorption, the compound then must penetrate the stratum corneum, diffuse into and through the viable epidermis into the dermis, and, finally, gain access to the systemic compartment through the vascular system. In addition, the substance may diffuse through the dermal and hypodermal layers to reach underlying tissues. As summarized in Table 215-1, within each compartment, the compound may diffuse down along its concentration gradient, bind to specific components, or be metabolized. The size or characteristics of each compartment may alter with time, and the factors determining diffusion within each compartment may be affected by disease state as well as the nature or the pharmacologic/biologic activity of the drug or its excipients.
Surface Applications of Formulations
Formulations differ in their physicochemical properties, and, as discussed in Section “Formulations,” this influences the kinetics of release and/or absorption. However, the principal consideration is that topically applied drug products represent a physically small compartment, which limits the amount of compound that can be applied to the skin surface. When a patient applies a dermatologic preparation, the layer of a formulation covering the skin is very thin (approximately 0.5–2.0 mg/cm2). Thicker layers are felt as “unpleasant” and are consciously or subconsciously rubbed off or spread to larger surfaces. This restricts the amount of an active compound that can effectively come in contact with the skin surface to approximately 5–20 μg/cm2 for a 1% (wt/wt) topical formulation.
However, even after being rubbed in, formulations do not remain homogeneous over the time course of penetration. For example, topical applications containing water, alcohol, or similar solvents undergo rapid evaporation.5 This phenomenon is readily recognized by patients as a cooling sensation. The evaporation results in rapidly increasing concentrations of nonvolatile substances on the skin surface, which may result in the formation of supersaturated “solutions” or, alternatively, precipitation of active ingredients. Formulations may also mix with skin-surface lipids or undergo time-dependent changes in their composition as excipients and drugs undergo absorption. Altogether, these considerations suggest that dramatic changes in the composition and structure of formulations may occur following surface application, all of which may determine the subsequent bioavailability of active ingredients.
The reservoir function was first described by Vickers,6 who observed that simple occlusion leads to the renewed onset of a glucocorticoid-mediated vasoconstriction several hours after it had declined. He interpreted this effect as renewed liberation of the glucocorticoid from a “reservoir” stored in the upper skin layers.
We define as reservoir the amount of an active ingredient that is still in contact with the nonvolatile constituents of its formulation after the latter had been massaged into the skin surface. The compound has not yet penetrated, but it cannot be removed by simple rubbing or contact with clothing or other tissues. The reservoir thus adheres to the skin surface and resides in the wrinkles and the upper layers of the stratum corneum. Reservoirs on eczematous skin may become even more prominent because of the scaliness of the skin. Recently, we discovered that the upper volume of the follicular channels serves also as a reservoir, which may result in a relative increase in absorption through appendages. In-vivo laser scanning microscopy measurements found that the hair follicles represent an efficient reservoir for topically applied formulations, which can be compared with the reservoir of the stratum corneum on several body sites.7,8 This phenomenon may be increased in formulations that contain particles or precipitates, given the evidence that appropriately sized particles can rapidly penetrate along the shafts of hair follicles to a depth of up to 100–500 μm.9–12
The optimum size of the particles for penetration into hair follicles is between 300 and 600 nm, which corresponds to the cuticular structure of the hairs.13,14 It was assumed that the rigid hair shaft acts as a geared pump, because this effect could only be observed in the case of moving hairs.14 The follicular reservoir may result in a relative increase in the absorption of topically applied substances. No evidence has been found that topically applied substances penetrate efficiently into the sweat glands. This may be due to sweat outflow or other, unknown reasons.
Formulations can be differentiated on the basis of whether they are designed to remain on the skin surface (sunscreen products and cosmetics), to be delivered to compartments in the skin (topical formulations), or to migrate across the skin into the central compartment (transdermal formulations).
Formulations may affect the kinetics and the degree of percutaneous absorption and, subsequently, the onset, duration, and extent of a biologic response. In the context of percutaneous absorption, there are several different parameters that should be considered when selecting a formulation1,15: the thermodynamic activity of the active ingredient16; the amount of compound that can be incorporated into the formulation17; the stability of the formulation on the skin surface (e.g., emulsions may break easily)18; the partition coefficient of the active ingredient between the vehicle and the stratum corneum19; and the enhancer activity.
In general, percutaneous absorption is proportional to the thermodynamic activity of the compound. Thus, the greatest flux is observed at the active ingredient's maximum solubility in a vehicle. Vehicles that are very good solvents should be avoided because they may retain the active ingredient on the skin surface.
Liposomes as Transdermal Delivery Systems
Liposomes are microscopic spheres comprising a bilayer that encloses an inner aqueous core. A wide variety of cosmetics contain liposomes. Liposome-based formulations have proved to be safe, cosmetically attractive, and well accepted. There is considerable evidence that, at least for some preparations, application of liposomes is mildly occlusive and improves the hydration level of the stratum corneum. Interest in the use of liposomes to enhance the delivery of drugs across the skin has been spurred by several observations in animal models: liposome formulations were believed to enhance the penetration of compounds across the skin or to optimize the retention of bioactive compounds in target tissues.20 However, these early studies, which relied largely on animal models, were followed by relatively few in-vivo studies for humans17 conducted under standard conditions.
The action mechanism of liposomes is based on a partly damaged liquid layer of the stratum corneum, so that the liposomes can penetrate efficiently into the skin barrier. Deep in the stratum corneum, the liposomes get damaged and release their drug, which has to pass through the last cell layers of the stratum corneum by itself to reach the living cells.21
There is no clear evidence that liposomes can pass the skin barrier as intact structures, but intact liposomes can penetrate along the hair shaft and this route may be appropriate for delivery of bioactive compounds into sebaceous glands or hair follicles.7,8 Rigid liposomes penetrate better into the hair follicles than flexible liposomes, which supports the assumption that the moving hairs act as a geared pump.21
The primary compartment that limits the percutaneous absorption of compounds is the stratum corneum. This thin (10–20 μm) layer effectively surrounds the body and represents a highly differentiated structure that determines the diffusion of compounds across the skin. The physical description of the stratum corneum is well documented,22 and it can be accurately described as “bricks” of bundled, water-insoluble proteins, embedded in a “mortar” of intercellular lipid.
The stratum corneum is a highly organized, differentiated structure. To participate fully in forming an effective barrier to diffusion, the biogenesis of the corneocytes as well as the synthesis and processing of the intercellular lipid must proceed in an orderly manner. Disruption in the kinetics of skin barrier formation by accelerating the division of the keratinocytes in the underlying layers will lead to a disruption in the barrier properties of the skin.23 Thus, the concept of dead or dying skin forming a passive barrier to diffusion is now replaced by a model of the stratum corneum as a highly differentiated structure with unique properties that are particularly suited for its role in forming the skin barrier (see Chapter 47).24
A variety of appendages penetrate the stratum corneum and epidermis, facilitating thermal control and providing a protective covering. Appendages are potential sites of discontinuity in the integrity of the skin barrier. The density of the hair follicles varies on different body sites. Hair follicles represent a reservoir that may store topically applied substances. A detailed analysis of the reservoir of the hair follicles showed that the highest reservoir is on the scalp, followed by the forehead and the calf.25 On the forehead, there are a high number of small follicles, while the calf contains fewer but larger hair follicles. These reservoirs are comparable to the reservoir of the stratum corneum on these body sites. The percentage of the hair follicles on the total skin surface varies between 0.2% and 1.3%, depending on the body site.25 Differences in the follicular penetration were observed in different ethnic groups.26 Hair follicles appear to present an important pathway for percutaneous absorption in nondiseased skin.11,12 This can be explained by the fact that only the upper wall of the follicular apparatus (the acroinfundibulum) is protected by a coherent stratum corneum, whereas in the lower part (infrainfundibulum), the corneocytes appear undifferentiated, and protection is incomplete, if not absent. Even solid particles may enter deep into the follicular orifice,9,10,22 a phenomenon that lends itself to the concept of follicular targeting of drugs.22
It follows that in relationship to the integral protection against the passage of xenobiotics in general, and drugs specifically, the barrier function of the interfollicular stratum corneum is even more potent than previously believed, whereas more research is needed relative to the follicular pathway. Recent investigations hint to the presence of active follicles (open to penetration) and passive ones.11
In principle, three penetration pathways are possible: (1) the intercellular penetration, inside the lipid layers around the corneocytes; (2) the follicular penetration; and (3) theintracellular penetration. Although in the past, the transcorneal penetration was assumed to be the only penetration pathway, recent investigations, as cited above in Appendages, have demonstrated that penetration via the hair follicles should be taken into consideration.25–27
Up to the present time, no evidence is available that topically applied substances pass the skin barriers by means of the intracellular route.
Pathways Across the Stratum Corneum
Several studies have directly visualized penetration pathways across the stratum corneum by electron microscopy. For example, osmium vapor can be used to precipitate n-butanol that has penetrated the stratum corneum.28 After a brief (5–60 s) exposure of murine or human stratum corneum, the alcohol was found to be enriched in the intercellular spaces (threefold), although significant levels were also found in the corneocytes. By using a different approach that involved rapid freezing, water, ethanol, and cholesterol were also found preferentially concentrated in the intercellular lipid spaces.29 Similarly, the penetration of mercury chloride through the intercellular lipid can be detected following precipitation with ammonium sulfide vapor.30
However, in most of these investigations, there was also significant localization of compounds in the corneocytes, more prevalent in the upper layers (stratum disjunctum). Thus, corneocytes undergoing desquamation appear to be relatively permeable, even to rather bulky ions such as mercury. There is additional evidence that other compounds can and do penetrate corneocytes. It is well established, for example, that occlusion or immersion of skin in a bath leads to swelling of the corneocytes, consistent with the entry of water. Other compounds have also been localized in corneocytes, such as the anionic surfactants that are bound to keratins. Low-molecular-weight moisturizers such as glycerol are likely to partition into the corneocytes and alter their water-binding capacity. Thus, the penetration of compounds into corneocytes cannot be excluded from considerations of percutaneous absorption pathways. The relevance of this step relates to whether it is rate determining, i.e., whether the diffusion of compounds within the intercellular lipid is restricted by the corneocytes.
Pathways Across the Hair Follicles
Using the method of differential stripping—a combination of tape stripping with cyanoacrylate surface biopsies—the amount of formulation stored in the hair follicles can be quantified.31 It was found that nanoparticles were stored 10 times longer in the hair follicles than in the stratum corneum.14 It should be noted that when topically applied substances penetrate into the hair follicles, they do not necessarily penetrate through the skin barrier into the living tissue, because the hair follicles also have barrier properties.
On the other hand, the particles can be used as efficient carrier systems for drug delivery into the hair follicles. The hair follicles represent an important target structure because they are surrounded by a close network of blood capillaries. Additionally, they are hosting the stem and dendritic cells which are important for regenerative medicine and monomodulation.
For optimal action, the drug should be released from the particles after having penetrated deep into the hair follicles. The pharmacokinetics is determined mainly by the process of the drug release from the particles in this case.61
In the past, there have been several attempts to detect follicular penetration.32–34 Experiments were performed on animal and human skin, with different densities of the hair follicles. Unfortunately, in all cases, the properties of the stratum corneum had also changed.
The analysis of the follicular penetration became possible after the development of a method that artificially closes the hair follicles in vivo.35 Using this method, it was demonstrated that the small molecules, such as caffeine, may penetrate through the skin barrier not only by the transcorneal, but also by the follicular routes.36
Inter- and Intraindividual Variation in Skin Barrier Function
Finally, it is worthwhile to consider the level of inter- and intraindividual variation in skin barrier activity, including that of follicles. The most accurate and reproducible measurement of skin barrier activity is transepidermal water loss.37 The extent of variation of this parameter for the same individual is estimated to be 8% by site and 21% according to the day of measurement. The variations between individuals are reported to be somewhat larger, ranging from 35%–48%.38 There appear to be no significant gender- or ethnic-dependent differences in skin barrier activity. The skin barrier activity of premature babies39 is markedly impaired, although skin barrier function appears normal for full-term infants. There seems to be no significant alteration in skin barrier activity as a function of age. Differences in skin barrier activity among different sites have been observed; barrier function can be ranked as arm ∼ abdomen > postauricular > forehead.37
Although the primary barrier to percutaneous absorption lies within the stratum corneum, diffusion within the viable tissue, as well as metabolism and resorption, also influence the bioavailability of compounds in specific skin compartments. These processes are interrelated, and factors that increase the rate of one of these processes inevitably influence the others. Because the development of dermatologic formulations is often focused on “targeted” delivery to living tissues, the manipulation of these processes offers a clear-cut rationale for increasing the therapeutic efficacy of dermatologic drugs.
The passage of compounds from the stratum corneum into the viable epidermis results in a substantial dilution. This is not only the relatively larger volume of the epidermis as compared with that of the stratum corneum, but also the lower resistance to diffusion within viable tissues, approximately corresponding to that of an aqueous protein gel.38 Drug concentrations of 10−4–10−6 M may be attained in the epidermis and dermis for substances that permeate readily. Although the actual concentration gradient of a compound is affected both by the physicochemical properties of the compounds as well as by the duration of application, a concentration gradient is present at all times. In other words, strategies to enhance percutaneous absorption generally result in a relatively uniform and parallel increase in the concentration of compounds in all compartments.