In comparison to other tissues, the stratum corneum intercellular spaces occupy a large volume fraction: 10% of the total volume ver-sus 0.5-1.5% in other tissues [26]. It reveals the important role of these lipids, which is confirmed by the high level of lipid synthesis in the epidermis which, although making up only 10% of the skin's mass, accounts for 25-30% of the total cutaneous activity (which corresponds to 20-25% of total body lipid synthesis) [27].
The composition of stratum corneum lipids is unique (Table II), since unlike other bio-logical membranes, they contain almost no phospholipids, but contain a large amount of ceramides and almost the same amount of cholesterol derivatives [28]. Six classes of ceramides, designated from 1 to 6 (Figure 13) have been isolated and identified in human stratum corneum [8]. Their structures contain long-chain bases, N-acetylated by different fatty acids.
These lipids are organized in a mixture of compact structure (Figure 14), and are ar-ranged to form lamellar bilayers (Figure 15), whose morphology has been widely
Figure 11: Typical profile obtained by sequentially tape stripping a constant area of skin on the flexor forearm of one subject, showing decrease of corneo-cyte release from the upper layers to the deepers.
From [25].
Figure 12: Effect of tape stripping on removed stra-tum corneum volume (µg/cell layer/cm2) and thick-ness (µm/cell layer). From [13].
investigated using electron microscopy [4]
and whose molecular ultrastructure has been studied using x-ray diffraction [30]. Because of its long chain, ceramide 1, an acylcera-mide, may function as a "molecular rivet", stabilizing the intercellular lipid lamellae.
There is also strong evidence to indicate that the intercellular lamellae are further stabi-lized by the chemical links between the long-chain ceramides and glutamate residues on the corneocyte protein envelope [24].
A lipid mixture would be expected to have a lower melting point than the respective pure lipids. However, the stratum corneum lipid phase, which consists of a great number of components, shows endothermic phase transi-tions at the relatively high temperatures of 75°C and 85°C (Figure 16) as measured by differential scanning calorimetry [31,32].
These were interpreted as melting proc-esses or as transitions from the
liquid-Figure 14: Model for the structure of bilayer intercel-lular stratum corneum lipids. From [35].
Figure 13: Representative structures of the six main fractions of human stratum corneum ceramides. From [8].
Table II: Composition of human stra-tum corneum lipids in weight percent-age. From [29].
Lipid % (w/w)
Cholesterol derivatives 38.8
Cholesterol 26.9
Cholesterol esters 10.1 Cholesterol sulfate 1.9
Ceramides 41.1
Ceramide 1 3.2
Ceramide 2 8.9
Ceramide 3 4.9
Ceramide 4 6.1
Ceramide 5 5.7
Ceramide 6 12.3
Fatty acids 9.1
Others 11.1
Figure 15: Model for the packed arrangement of
inter-crystalline to the isotropic state. These high melting temperatures indicate that the lipids of the stratum corneum are arranged with a high degree of order and in a high density.
Lower transitions have been identified at 40°C, and were initially attributed to melting of sebaceous lipids. It has been recently shown that they rather represent a
solid-to-fluid phase change for a discrete subset of stratum corneum lipids [33]. Thus, intercellu-lar lipids coexist in different physical states:
crystalline, gel, and liquid [34].
This highly ordered and stable lipid struc-ture, together with the previously mentioned tortuous architecture of the corneocytes is responsible for the effective barrier to both penetration of exogenous chemical com-pounds and water loss from the epidermis.
However, extensive infrared spectroscopic studies have shown that chemical compounds such as penetration enhancers are likely to perturb this structure by increasing the lipid disorder (Figure 17) thereby reducing the dif-fusional resistance of the barrier membrane.
Evidence of this effect was shown, among others, for oleic acid [38,39] and other fatty acids [40], ethanol [41], Azone® [42], and phospholipids [43].
Figure 16: Differential scanning calorimetric profile of human stratum corneum, showing the characteristic endothermic phase transitions associated to melting of sebaceous lipids (T1=40°C), melting of bilayer lipids (T2=75°C and T3=85°C), and protein denaturation of intracelllar keratin (T4=107°C). From [37].
Figure 17: Model for the penetration enhancer induced fluidization of the intercellular lipids. Small and polar mole-cules such as ethanol are believed to affect the polar heads of the lipids, whereas lipophilic compounds such as oleic acid, other fatty acids, phospholipids, and Azone® are likely to insert into the alkyl chains. From [35].
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In Vivo Methods for the Assessment of Topical Drug Bioavailability
Ingo Alberti,1,2 Yogeshvar N. Kalia,1,2 Richard H. Guy1,2,3
1Centre Interuniversitaire de Recherche et d'Enseignement "Pharmapeptides", Campus universitaire, F-74166 Archamps (France); 2Laboratoire de Pharmacie Galénique, Section de Pharmacie, University of Geneva, CH-1211 Genève 4 (Switzerland); 3To whom correspondence should be ad-dressed.
Published as part of review paper in Pharmaceutical Research 2008, Vol. 25, No. 1, 87-103.
Abstract
This paper reviews some current methods for the in vivo assessment of local cutaneous bioavailability in humans af-ter topical drug application. Afaf-ter an introduction discussing the widespread use of generic dermatological products and the limited in vivo methodologies available for bioequivalence assessment, the focus turns to the relevance of studies on human subjects and the definition of local bioavailability. The available techniques are then reviewed in detail, with particular emphasis on the tape stripping methodology, which is currently the subject of much debate. Two approaches are discussed: the assessment of local drug bioavailability from (i) the removed tape-strips and (ii) the tape-stripped skin. A very recent and promising dermatokinetic technique, microdialysis, is also described. Other techniques of lim-ited use such as the skin biopsy, the suction blister, and the follicle removal technique are also mentioned.
Keywords: Cutaneous bioavailability; Cutaneous drug concentration; Tape stripping; Skin microdialysis; Skin biopsy;
Suction blistering; Dermatopharmacokinetics.
Introduction
The generics' appeal
he escalating cost of drugs is one of the principal reasons for the wide-spread use of generic drugs, and has been encouraged by the relaxation of regula-tory requirements. Topical dermatological formulations are obviously subject to the same concerns, the most marketed generics in this field including corticosteroids, antiacneic, antifungal, antibiotic and antiviral agents. A general consequence, is that during the last two decades most countries have brought in
legislation adapted to generics. The purpose of these regulations is to offer more economic drugs to the market, by eliminating the costly and possibly unnecessary requirements for duplicate safety and efficacy studies. Manu-facturers have to document pharmaceutic and therapeutic equivalence (bioequivalence) of the generic drug in relation to the original:
any shorter and cheaper procedure will thereby allow the approval process to be ac-celerated. In reality, pharmaceutical equiva-lence methods are much easier to implement than bioequivalence methods, since they may be realized in a completely controlled
envi-T
ronment (i.e., where all the physicochemical parameters, such as temperature, concentra-tion, and pH are known). In contrast, bio-equivalence methods, when applied to human beings, are more difficult and complicated to carry out in a controlled fashion, since the environmental and intrinsic parameters (e.g., the individual pharmacological response) are often not constant or not known. This obvi-ously makes difficult the interpretation of the results, since a high degree of variability im-plies, for a study to reach a sufficient statisti-cal certainty, that the number of subjects be increased, which consequently raises the cost of the study.
Furthermore, each class of therapeutic agents needs a particular methodology for the bioavailability/bioequivalence assessment, which additionally complicates the proce-dures of drug development. Currently, two main strategies are used and accepted: the first is indirect, and concerns pharmacody-namic measurements of the pharmacological response to the drug within the skin, for ex-ample the induced vasoconstriction effect of corticosteroids [1], or the erythema-induced effect of nicotinates for the evaluation of non-steroidal anti-inflammatory drugs [2]. Yet, this strategy is somewhat arbitrary, since those are surrogate effects which are related to the primary pharmacological effect but do not strictly characterize the real efficacy of the drug.
For drugs which do not exert any measur-able pharmacological in vivo effect, a second and more direct strategy is available, which involves pharmacokinetics measurement of the drug concentration in the skin itself. In vivo methods concerning this strategy will be discussed in this paper, since they appear to be more universal, and governmental regula-tory agencies are interested in promoting them, since they could be implemented with respect to a large array of drugs, and will pos-sibly limit expensive and time-consuming comparative clinical trials to assess bio-equivalence of a generic drug.
Relevance of studies on human subjects In order to approve original or generic topical dermatological products, the regula-tory administrations require bioavailabil-ity/bioequivalence studies. For example, in the U.S., the FDA also demands, in vivo stud-ies along with in vitro studstud-ies, for original products and for generics based on post-1962 approved drugs [3,4]. This is not surprising, since even if in vitro studies on human skin may give an estimate of the penetration be-haviour of a drug at an early stage of devel-opment, they remain a surrogate predictor of drug performance in vivo. This is not to say that in vitro testing should be skipped: its use-fulness remains obvious for comparative
pharmaceutical evaluations of formulations containing original or generic drugs.
However, the data preference for in vivo bioavailability/bioequivalence, is due to the lack of a satisfying correlation between in vivo and in vitro models [5], since in vitro techniques subject the skin to conditions not seen in vivo or in clinical application: absence of dermal blood clearance, overhydration of the skin layers, degradation of the tissue and modification of the metabolic activity [6]. In particular, for lipophilic molecules (log [oc-tanol/water partitioning] values bigger than 3), the presence of the aqueous dermis in vitro acts as an artificial barrier retaining those permeants, whereas in vivo they are more ef-ficiently removed by the dense capillary net-work immediately below the dermo-epidermal junction.
Furthermore, the in vitro conditions lead to a higher variability of skin permeability than in vivo. In a comprehensive comparison of a number of data sets, Southwell and co-workers [7] showed that intra-specimen vari-ability was higher in vitro (43%) than in vivo (27%), as well as inter-specimen variability, which was found to be higher in vitro (66%) than in vivo (45%).
However, local cutaneous bioavailabil-ity/bioequivalence studies performed on hu-man subjects have some drawbacks: they are very expensive and time-consuming, both in the pre-clinical and clinical stages of devel-opment of an original or generic drug. In
ad-dition, many in vivo methods are more or less invasive, so that animal models are sometimes preferred. This could explain why, except for clinical trials, methods for in vivo local cuta-neous bioavailability/bioequiva-lence assess-ment are relatively difficult to find in the lit-erature, even if in the last decade less invasive (or less painful) approaches have been devel-oped, as will be reported in this paper.
Assessment of local bioavailability/
bioequivalence in cutaneous drug delivery The effectiveness of a topical dermatologi-cal formulation is usually evaluated, in vivo, in terms of the cutaneous bioavailability of the drug, which is a regulatory requirement for marketing approval, and which is used as a basis for bioequivalence comparisons.
Strictly speaking, bioavailability is defined as the measure of both the true and total amount of drug reaching the general circulation from an administered dosage form [8]. The concept was originally intended for oral drugs, de-signed for exerting a systemic effect, and therefore releasing the active moiety in the blood at a significant rate and extent, thus generating measurable levels. It was then ex-tended to other dosage forms generating con-sistent and sustained drug blood levels, such as transdermal delivery devices, which are locally applied, but are intended to release the drug in the systemic circulation and treat dis-eases located far from the skin. They have
consequently also been adapted to topical dermatological formulations (such as creams, ointments, gels, lotions, etc.), which are though intended to act locally and not sys-temically. For those dosage forms, the classi-cal concept of bioavailability must be recon-sidered, since many specific features of topi-cal cutaneous delivery may no longer be per-tinent, and consequently systemic bioavail-ability may not properly reflect local cutane-ous bioavailability.
The first specific feature, is that even if there is an evident relationship between the applied drug amount and the generated sys-temic blood levels [9], the latter are often too low to be detectable by conventional analyti-cal methods [10]. Radiolabeled molecules may obviously overcome this problem, but ethical concerns preclude these kinds of in vivo investigations in most countries, and in addition, when total radioactivity is monitored drug and labeled metabolites are not distin-guished. The reason why such low blood lev-els are generated, is that only a thin layer of formulation is applied to the skin, and fur-thermore, over a small surface area. For creams and ointments, the amount of formula-tion is generally less than ten milligrams per square centimeter (typically 2-3 mg/cm2).
This usually contains a drug dose of two or-ders of magnitude below (typically some per-cent), which means some tens of micrograms drug per square centimeter of skin surface.
Such a small and finite dose is generally not
enough to generate significant blood levels, for the chemical potential providing the driv-ing force of penetration (the thermodynamic activity of the drug) is rapidly exhausted [11].
Furthermore, physico-chemical modifications of the freshly applied formulation, e.g., evaporation of solvents [12], as well as the cutaneous metabolic actvity [13], could addi-tionally reduce the low amount reaching the dermal capillaries.
The second specific feature of topical de-rmatological formulations is that the vehicle plays a decisive role in the local absorption of the drug. Although drug release from the for-mulation and penetration into the skin are subject to the same physico-chemical princi-ples in both topical cutaneous and transdermal
The second specific feature of topical de-rmatological formulations is that the vehicle plays a decisive role in the local absorption of the drug. Although drug release from the for-mulation and penetration into the skin are subject to the same physico-chemical princi-ples in both topical cutaneous and transdermal