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The human skin protects the body from physical, mechanical, and chemical insults while preventing endogenous water loss. This function is predominantly achieved by a thin (10–30 µm) cornified outermost layer-the stratum corneum (SC)-generated through terminal differentiation of the basal epidermal keratinocytes. The stratum corneum protects the human body, but also severely limits drug delivery into and across the skin.
The human skin protects the body from physical, mechanical, and chemical insults while preventing endogenous water loss. This function is predominantly achieved by a thin (10–30 µm) cornified outermost layer-the stratum corneum (SC)-generated through terminal differentiation of the basal epidermal keratinocytes. The stratum corneum protects the human body, but also severely limits drug delivery into and across the skin. The historical and theoretical understanding of the type of compound that will permeate the skin is based on the “500-dalton rule” (1, 2), where it is assumed that most compounds permeating the SC have a relatively small molecular weight (<500 Da) and are moderately lipophilic (log P 1–3.5) (3). Such an understanding was, however, based on the assumption of transdermal delivery across healthy skin with an intact barrier. Yet, according to the data from Citeline’s Pipeline database, 7% of topical medicines for the treatment of skin disease contain drugs with molecular weights from 600–1000 Da. Tacrolimus and pimecrolimus (804 Da and 810 Da, respectively), for example, are the two most well-known compounds that appear to contradict the “500-dalton rule.” Nevertheless, at present, there is no approved topical formulation containing a drug with a molecular weight of more than 1000 Da.
The literature consists of publications claiming topical delivery of macromolecules. Why the discrepancy? Critical evaluation of this literature suggests that some of the methodologies can be misleading and may contain artifacts that make translating to a clinical significance difficult. One of the challenges when attempting to understand passive topical delivery of compounds is establishing a model or testing system that mimics the in-vivo condition. This task is even more challenging when the size of the compounds exceeds 1000 Da because many of the analytical tools used to detect and quantify small molecules are problematic for larger molecular weight compounds. Many researchers have used animal models, such as rodents and/or minipigs, either in vitro or in vivo, to assess the passive delivery of large molecules. It can be argued that these models have yet to show good correlation to the clinical situation as proven by the lack of products on the market. In fact, many of these studies have been shown to be misleading as a result of inappropriate experimental design or a lack of contextualization of this limitation in the study conclusions.
There have been, nonetheless, some glimmers of hope when polysaccharides, proteins, and oligonucleotides are explored. Hyaluronic acid (HA), a naturally occurring polyanionic polysaccharide up to 1000 kDa, is found in the skin and has been shown to be a key molecule involved in skin moisture due to its capacity to retain water (4). There have been many publications investigating the topical delivery of HA. One of the earliest and most comprehensive studies examined the topical delivery of HA following application to the human forearm in situ (5). Tritiated HA was detected in the dermis just below the epidermis, and the authors took many steps to eliminate artifacts to ensure the observations were real and valid. Since publication in 1999, the group did not report further findings on HA. However, there have been many other studies claiming to have shown HA permeation through human skin, yet the topical delivery HA continues to be taken with scepticism (6–11).
In addition, latex proteins (14-52 kDa) have been proposed to cause hypersensitivity allergen-based reactions (12). Latex proteins are larger molecular weight compounds (14–52 kDa); hence, it is interesting how these proteins can produce allergic responses in the skin. It is presumed that the proteins are able to cross the skin barrier to elicit the response. In fact, there are studies that show latex proteins were able to penetrate excised human skin, and that exposure induced an IgG1 response in vivo (13).
There have been several intriguing studies exploring the topical delivery of oligonucleotides within the past decade. Experimental studies using a nuclear factor kappa B decoy oligonucleotide (13 kDa) showed initial promise as a potential topical treatment for atopic dermatitis. The product progressed as Avrina into Phase1/2 (14) but despite the clinically positive data, it does not seem to have progressed further. Some of the constraints related to modifications of antisense molecules and their fixed structural nature may explain this lack of progression. More recently, topical application of a tumor necrosis factor (TNF)-α suppressing antisense spherical nucleic acid showed that treatment with the highest dose resulted in a statistically significant decrease in TNF messenger ribonucleic acid (mRNA) expression in psoriatic skin (15). This technology will be interesting to watch because it appears there are several similar compounds in early development.
Aptamers are a subclass of large molecules that have been shown to have high binding affinity and selectivity with the ability to disrupt protein–protein interactions. Currently, it is not possible to disrupt these protein–protein interactions with traditional small molecules. Aptamers, therefore, represent a new class of molecules that could have antibody-like binding affinity with the possibility of topical delivery. Evidence from the literature supports their rapid clearance from the systemic circulation, thus limiting unwanted systemic side effects and restricting the biologic effects of topically administered aptamers to the skin (16). Interestingly, aptamers offer significant conformational plasticity and flexibility. Moreover, their structure can be modified without the loss of significant activity (17, 18).
In a recent publication, researchers at GlaxoSmithKline, University of Reading, and MedPharm showed, for the first time, that a 62-nucleotide (20,395 Da) RNA-based aptamer, highly specific to the human interleukin (IL)-23 cytokine, permeated intact human skin to therapeutically relevant levels in both the epidermis and dermis (19). This observation was particularly surprising considering the compound was 40 times larger than what is commonly accepted as possible for passive topical delivery in the skin.
In the study, the authors used multiple approaches to demonstrate the topical delivery of the aptamers, including fluorescently labeled aptamer, confocal microscopy, and a novel dual hybridization assay that used capture and detection probes with oligonucleotide precipitation to be able to quantify the aptamer at picomolar levels. They showed the IL-23 aptamer delivered into the skin was significantly above the cellular IC50 values (119,000-fold > IC50 in the epidermis; 3400-fold > IC50 in the dermis) when treated topically using a simple cream formulation. This portion of the study used freshly excised human skin and a diffusion cell commonly referred to in-vitro penetration/permeation (IVPT) and is considered the “gold standard” for assessing topical delivery for both biopharmaceutical companies and regulatory agencies. To confirm the IVPT delivery and to help visualize this delivery, confocal microscopy was introduced on sections from the IVPT study, and this aptamer appeared to localize to the intracellular and extracellular compartments within the viable epidermis (see Figure 1). It was interesting that this observation was noted to confirm a previous publication, showing the uptake of a different aptamer into primary human keratinocytes (20). From this independent observation, it could be extrapolated that intracellular and extracellular targets are possible with this technology.
To ensure the delivery observed with the other techniques was at therapeutic levels and the aptamer was bioavailable, the authors developed a Th17 mediated biological model using ex vivo human skin and showed the IL-23 aptamer was able to suppress IL-17 and IL-22 mRNA production (see Figure 2) following topical application. Interestingly, there may be some structural commonalities between HAs, latex allergens, oligonucleotides, and aptamers, which potentially explain the positive observations for topical delivery; however, this hypothesis requires further investigation. Nevertheless, if this in-vitro work were to be confirmed clinically, this result could present a major breakthrough in dermatology and topical drug delivery as it could open new areas of research and potentially targets that are not accessible using traditional small molecules.
There are several critical experimental parameters required to ensure robust, artifact-free results to allow for improved translation to clinical situations. Some of these experimental parameters are the use of human skin with an intact or uncompromised barrier, clinically relevant dosing volumes, a validated highly sensitive analytical method for extraction and quantitation of the compound, and potentially, confirmation of biological activity and structure using human skin.
The first step is to develop an analytical method to ensure the detection, analysis, and quantitation of the macromolecule is fit for purpose and free from interference. Having duplicate analytical methods using alternate techniques is an ideal approach to further confirm that the observations are valid. As with any topical program, it is imperative that proper preformulation studies occur to establish compound and formulation stability both from a chemical and biological activity sense. The next step is to screen compounds and formulations for passive topical delivery using human skin in vitro. The final and perhaps most crucial step is to ensure the macromolecule is tested for biological activity ideally using ex-vivo skin to assess target engagement from a topical application. Given the counterintuitive challenge of proving topical delivery with macromolecules, study designs tend to include a majority of controls (both negative and positive) to ensure an unbiased, non-artifact, and robust result.
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Supplement: APIs, Excipients, & Manufacturing 2018
When referring to this article, please cite it as M. Brown and J. Lenn, "A Perspective on the Topical Delivery of Macromolecules,"Pharmaceutical Technology APIs, Excipients, & Manufacturing 2018 (September 2018).
Marc Brown, PhD, is chief scientific officer and co-founder of MedPharm; and Jon Lenn, PhD, is senior vice-president for MedPharm’s US operations.