Figure 3 – Skin Drug Delivery. A) Pathways
into the skin for transdermal drug delivery: a) Transcellular pathway
(penetration through the corneocytes); b) Intercellular pathway
(penetration between the corneocytes through the intercellular lipids);
c) Intrafollicular pathway (penetration through the hair follicles); d)
Polar pathway (penetration through the polar pores); adapted from (Sofia
A. Costa Lima., 2018); B) Types of drugs entrance routes
through the skin.
The intercellular pathway involves the passage of the drugs through the
lipid matrix that occupies the intercellular spaces of the corneocytes
and is usually the preferred route for lipophilic substances. Otherwise,
the transcellular pathway, also known as the intracellular pathway,
occurs through the successive skin layers and dead cells and allows the
transport of hydrophilic or polar substances. The transappendageal
pathway uses the different skin appendages to enter through the skin.
Various sweat glands, hair follicles and pores opening to the outer
surface of the skin via their ducts can be used as a possible way for
the entrance of drugs. These polar pores are located between cells and
encircled by polar lipids, which make small holes in SC (Alkilani
et al., 2015, Sofia A. Costa Lima., 2018). Hence, it was considered an
inessential pathway for drug penetration but nowadays, current research
suggests that hair follicles and sweat glands may present an alternate
pathway for a diffusing molecule (Uchechi et al., 2014). In the polar
pathway, the penetration of the drugs occurs through the polar pores
available in the skin.
When the drugs are able to penetrate deep in the skin, from the surface
through the various layers, this type of penetration is called
“transdermal drug delivery” (TDD). The drug firstly pass through theSC and then permeates via the viable epidermis and dermis by
diffusion. After reaching the dermal layer, the drug becomes available
for the uptake into the systemic circulation (Alkilani et al., 2015).
TDD has advantages namely over hypodermic injections as the drugs
(usually administrated in patches) can be applied only one time and
released for a longer period of time (with no need of additional
applications), is almost pain-free and doesn’t lead to the generation of
dangerous medical waste such as needles and syringes. Furthermore,
transdermal devices can be self-administered, and the administration can
be easily stopped in case of need by removal of the patch (Van Gele et
al., 2011).
The different types of drugs entrance routes through the skin and their
classification are summarized in Figure 3B.
Healthy skin mimetic
models
Despite de fact that the in vivo human skin is the most realistic
and gold standard experimental model for the investigation of drugs
interaction with the skin, the use of this model is not always possible
mainly because of the ethical concerns, regulatory issues, laboratory
facilities and the potential risk associated to the eventual toxic
effects of the drugs (Van Gele et al., 2011). Moreover, the results
obtained by the use of human ex vivo models present significant
variability because samples are usually obtained from different
anatomical places of the same donor, different donors and have
unpredictable character depending on the different subjects or different
age groups (Flaten et al., 2015). These facts reinforce the need of
alternative models able to better mimic the real scenario of drug
interaction with the skin and concomitantly allowing reproducible
results (Abd et al., 2016).
In this section, an overview of the existent ex vivo and in
vitro mimetic skin models will be given.
Ex vivo human and
animal models
During long time, the main way for the preclinical research of new drugs
and for the optimization of topical drug formulations was the
investigation considering the use of ex vivo skin mimetic models.
The literatures describe two main groups of ex vivo models
obtained from human or animal organisms (see references (Abd et al.,
2016, Flaten et al., 2015) for reviews).
Human skin is absolutely the most suitable model for study TDD (Ruela et
al., 2016). The skin samples used in ex vivo permeation assays
can be obtained from different origins namely from plastic surgeries,
amputations or cadavers and in generally the skin excerpts can be
collected from different organs, such as the abdomen, back, leg or
breast (Schaefer et al., 2008). Different membrane types can be obtained
by using human skin excerpts for further use in drug permeation studies.
Full-thickness skin models, in which the excisions containing connective
tissue and subcutaneous fat and consists of all layers below, including
the dermis, are reported as useful model to test different drugs and
formulations (Abd et al., 2016, Cross et al., 2003, Manca et al., 2014,
Junyaprasert et al., 2012, Dragicevic-Curic et al., 2008,
Dragicevic-Curic et al., 2010, Elmoslemany et al., 2012, Bragagni et
al., 2012, Cal, 2006, Sahle et al., 2014, Gaur et al., 2013, Marimuthu
et al., 2012).
Ex vivo epidermal membranes models are also used for permeation
experiments and those models are obtained from thermal treatment of
full-thickness skin (immersion in hot water) (Junyaprasert et al., 2012,
Kligman and Christophers, 1963) or by chemical action namely by the use
of different reagents such as ethylenediaminetetraacetic acid, ammonia
and enzymes (Cross et al., 2003) in order to separate the membrane at
the dermal–epidermal junction. Other methodologies using human
dermatomed skin (Dragicevic-Curic et al., 2010, Dubey et al., 2007,
Clares et al., 2014, Marepally et al., 2013) or dermatopharmacokinetic
method in which tape stripping is used to remove SC layers have
been described (reviewed in (Abd et al., 2016)).
More recently, abdomen skin samples from patients who underwent
abdominoplasty are used as skin models (Ternullo et al., 2018). Many
examples report the use of human ex vivo skin models (reviewed in
references (Flaten et al., 2015, Abd et al., 2016)), as the
investigation of the dermal uptake and percutaneous penetration of some
organophosphate esters in a human skin ex vivo model (Frederiksen
et al., 2018). In another study, the effect of some nanoemulsions
containing alpha-tocopherol was evaluated in skin wounds either in cell
lines and using ex vivo human biopsies samples (Bonferoni et al.,
2018).
The use of skin perfusion models, a surgically prepared portion of skin
including subcutaneous fatty tissue with assured continuous vascular
circulation is reported (Ternullo et al., 2017a, Ternullo et al.,
2017b), to test different drugs, namely nanoparticle formulations. The
use of this model is considered a promising strategy since they present
benefits over in vitro models, as they overcome the existence of
only epidermis and part of the dermis and the lack of a vascular system
as verified in the most commonly used in vitro models (Ternullo
et al., 2017a, Ternullo et al., 2017b).
Regarding animal ex vivo models, pig skin models are the most
relevant because of the multiple anatomical, physiological and
histological similarities with the human skin such as the dermal/
epidermal thickness ratio, epidermal thickness, similarity in hair
follicle and blood vessel density in the skin and content of SCceramides, dermal collagen and elastin (Abd et al., 2016). The pig skin
is easily obtained as a waste from animals slaughtered for food. Amongst
the different parts of the pig body, the central outside part of the
porcine ear has been the mostly recommended due to the analogy with
human skin layers (Meyer et al., 2006). Variability of permeability in
different samples of pig skin also takes place. The pig ear skin
permeability is comparable with human skin. In fact, studies showed a
good correlation especially for lipophilic substances. Furthermore, the
age of the animal influences the permeability of the drugs, however most
of literature does not specify the age of animal (reviewed in (Flaten et
al., 2015)).
Many different drugs and formulations such as liposomes, nanoparticles
and microemulsions have been studied using ex vivo pig skin
models. Amongst the number of studies available, some reports describe
the evaluation of the permeation of liposomes containing different drugs
in excised pig ear (Scognamiglio et al., 2013, Knudsen et al., 2012,
Gillet et al., 2011). Other studies tested the permeation of different
nanoparticles in pig ear models (Gomes et al., 2014, Pople and Singh,
2011, Şenyiğit et al., 2010). Most recently, new formulations including
a transferosomal gel were tested using pig ear skin as an ex vivomodel for the study of the transdermal permeation and delivery of the
drug (Das et al., 2017). The use of excerpts from other pig skin
regions, namely from abdomen (Nagelreiter et al., 2013) and dorsum
(Hathout et al., 2010) is also described. Furthermore, newborn pig skin
excisions are used as skin models for evaluation of topical drug
formulations (Cilurzo et al., 2007).
In addition to the pig skin models, several other animals are used
namely primates, mice, rats, guinea pigs, rabbits, bovines (udder) and
snakes (shed skin). However, these models require ethical permissions.
Since 2009, the use of animals for collection of toxicological data for
cosmetic ingredients has been prohibited in the EU (76/768/EEC, February
2003) (Van Gele et al., 2011).
Mainly due to the fact that primate research is highly restricted and
very expensive, skin of rodents (mice, rat and guinea pigs) is sometimes
considered for permeation studies, due to its high availability, small
size and quite low price. There are available many hairless strains
which can be advantageous for this type of studies (Abd et al., 2016).
Amongst rodents, rat skin is most like human skin however many studies
pointed out the fact that its skin is more permeable than human skin
(Barber et al., 1992, Chowhan and Pritchard, 1978, Hughes and Edwards,
2010, Schmook et al., 2001, van Ravenzwaay and Leibold, 2004). Yet, a
study has shown that hairless mouse skin is an inadequate model for
assessing the effects of the skin penetration enhancers (Bond and Barry,
1988).
Shed snake skin was considering as well as a useful model to mimic human
skin and it can be obtained without killing the animal however it lacks
hair follicles (Rigg and Barry, 1990, Itoh et al., 1990, Wonglertnirant
et al., 2012, Kumpugdee-Vollrath et al., 2013). Additionally, udders
from slaughtered cows are used as an ex vivo animal model and
studies involving the comparison of this model with porcine skin has
confirmed that both models are well correlated, thus enabling its use
for studies regarding topical administration of drugs (Netzlaff et al.,
2006).
The several ex vivo animal models mainly differ in the thickness
of SC , hair density, number of corneocyte layers, hydration,
lipid profile and morphology which may constitutes several advantages
and limitations of each model. The most relevant features are summarized
in Table 1 (Flaten et al., 2015).