Studies and methodologies on vaginal drug permeation

Abstract

The vagina stands as an important alternative to the oral route for those systemic drugs that are poorly absorbed orally or are rapidly metabolized by the liver. Drug permeation through the vaginal tissue can be estimated by using in vitro, ex vivo and in vivo models. The latter ones, although more realistic, assume ethical and biological limitations due to animal handling. Therefore, in vitro and ex vivo models have been developed to predict drug absorption through the vagina while allowing for simultaneous toxicity and pathogenesis studies. This review focuses on available methodologies to study vaginal drug permeation discussing their advantages and drawbacks. The technical complexity, costs and the ethical issues of an available model, along with its accuracy and reproducibility will determine if it is valid and applicable. Therefore every model shall be evaluated, validated and standardized in order to allow for extrapolations and results presumption, and so improving vaginal drug research and stressing its benefits.
Copyright © 2015. Published by Elsevier B.V.

Full text

Studies and methodologies on vaginal drug permeation☆
Rita Monteiro Machado
a
, Ana Palmeira-de-Oliveira
a,b
, Carlos Gaspar
a,b
,
José Martinez-de-Oliveira
a,c
, Rita Palmeira-de-Oliveira
a,b,d,
⁎
a
CICS:UBI —Health Sciences Research Center, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal
b
Labfit, HPRD —Health Products Research and Development, Lda, Covilhã, Portugal
c
Child and Woman's Health Department, Centro Hospitalar Cova da Beira EPE, Covilhã, Portugal
d
Pharmacy Department, Centro Hospitalar Cova da Beira EPE, Covilhã, Portugal
abstractarticle info
Available online 16 February 2015
Keywords:
Vaginal drug delivery
Drug permeation
Vaginal permeability
Vaginal dosage forms
Franz cell systems
Ussing chambers
The vagina stands as an important alternative to the oral routefor those systemic drugsthat are poorly absorbed
orally or are rapidly metabolized by the liver. Drug permeation through the vaginal tissue can be estimated by
using in vitro,ex vivo and in vivo models. The latter ones, although more realistic, assume ethical and biological
limitations due to animal handling. Therefore, in vitro and ex vivo models have been developed to predict drug
absorption through the vagina while allowing for simultaneous toxicity and pathogenesis studies.
This review focuses on available methodologies to study vaginal drug permeation discussing their advantages
and drawbacks. The technical complexity, costs and the ethical issues of an available model, along with its
accuracy and reproducibility willdetermine if it is valid and applicable. Therefore every model shallbe evaluated,
validated and standardized in order to allow for extrapolations and results presumption, and so improving vag-
inal drug research and stressing its benefits.
© 2015 Elsevier B.V. All rights reserved.
Contents
1. Introduction...............................................................14
2. Vagina:anatomy,histologyandphysiology................................................. 15
3. Drugabsorptionfromthevagina......................................................15
4. Vaginaldrugpermeationmethodologies ..................................................16
4.1. In vitro models ..........................................................16
4.2. Ex vivo models...........................................................18
4.3. Permeationtests.......................................................... 21
4.3.1. Franzcells ........................................................ 21
4.3.2. Flowthroughdiffusioncells ................................................ 22
4.3.3. Ussingchambers .....................................................22
4.4. In vivo models...........................................................24
5. Conclusions ...............................................................24
References ..................................................................24
1. Introduction
Researchers are now devoted to find new forms or to re-discover safer
and more effective alternative routes for the administration of drugs that
are poorly absorbed orally or precociously suffer metabolization [1–3].
The vaginal route has been considered of great interest for drug delivery,
since it enables both local and systemic drug delivery [4,5], allowing
for the absorption of peptide and other macromolecules, and even
nanoparticles [6–8].
The vaginal route provides different advantages over the oral one but
it is not deprived of inconveniences [9,10]. Its large surface area, rich
blood supply, ability to bypass hepatic first-passage, avoidance of gastro-
intestinal side effects, and relatively high permeability to a wide range of
molecular weight drugs are some of its physiological characteristics that
Advanced Drug Delivery Reviews 92 (2015) 14–26
☆This review is part of the Advanced Drug Delivery Reviews theme issue on “Vaginal
Drug Delivery”.
⁎Corresponding author at: CICS–UB I —Health Sciences Research Center, Faculty of
Health Sciences, University of Beira Interior, Av. Infante D. Henrique, 6200–506 Covilhã,
Portugal. Tel.: +351 275329002; fax: +351 275329099.
E-mail address: rpo@fcsaude.ubi.pt (R. Palmeira-de-Oliveira).
http://dx.doi.org/10.1016/j.addr.2015.02.003
0169-409X/© 2015 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Advanced Drug Delivery Reviews
journal homepage: www.elsevier.com/locate/addr

contribute to its pharmacokinetic advantages [11–14]. However, drug ab-
sorption through the vagina may be affected by variations in epithelial
thickness and by changes in the vaginal milieu composition that occur
as a consequence of age dependent and cyclic physiological conditions
or sexual intercourse. Moreover, leakage and self-cleaning action of the
vaginal tract may reduce drug bioavailability [5,15]. Furthermore, general
disadvantages of vaginal drug delivery include its obvious gender speci-
ficity, cultural background limitations and personal hygienic care inter-
ference [12,14].
Vaginal drug delivery systems include solutions, semisolids (creams,
ointments and gels) and solid formulations (tampons, capsules, pessa-
ries, suppositories, films, sponges, powders and special controlled re-
lease devices like the intravaginal ring) as well as other types of
formulations such as aerosols and particulate systems integrated in ad-
equate drug delivery systems [16].Efficacy of drug delivery systems will
rely on their ability to promote adequate drug concentrations at the
targeted site of action. When a systemic effect is the objective through
this route, drugs must be transported across the epithelium to gain ac-
cess to dermal vessels and the systemic circulation [17]. On the other
hand, when a local effect is the goal, as is the case for some antimicro-
bials and microbicides, retention of the drug at the surface of the vagina
is desirable with low grade of absorption [18–21].
Drug permeation studies are mandatory for vaginal drug administra-
tion when systemic delivery is intended, and are important for safety
characterization when the objective is to limit the activity to the vaginal
wall surface or its contents. The in vitro models developed to predict
drug permeation not only provide information on absorption rates
and efficacy, but also help in investigating and understanding the path-
ogenesis of various microbiological diseases [22–24].
So, applicable and reproducible test assays are important to provide
comprehensiveness about mechanisms of permeation, absorption and
mode of action of active substances for vaginal application [25] and
also to characterize vaginal drug delivery systems and the formulation
ability to either promote or avoid permeation through the vagina [26,
27].
Previously published scientific studies on this subject are quite un-
clear concerning the specific meaning of permeability and permeation
terms. We assume that, by definition, permeability is the property of
membranes or barriers of an organ or structure of being permeable to
substances, while permeation denotes the ability of substances, like
drugs, to permeate through a membrane/barrier [28]. Nevertheless, it
is clear that this difference is not assumed uniformly and that the
terms “drug permeation”and “drug permeability”have been and still
are used interchangeably in the literature.
2. Vagina: anatomy, histology and physiology
The vagina is described as an expandable, longitudinally S-shaped,
fibromuscular, collapsed canal showing at transverse cross-section an
Hconfiguration, with the anterior and posterior walls contacting each
other in current conditions. It extends from the cervix of the uterus to
the vestibule [2,29,30], presenting approximately 7–10 cm in length,
more than 4 cm in width and 150–200 μm in thickness. The posterior
wall is longer than the anterior one, a consequence of the asymmetrical
position of the cervix at the vaginal vault [25,31–33].
The vagina is the female sexual organ by definition, and though it
normally does not harbor glands, it is usually referred as a mucosa. In
fact, despite not having a secreting role, the vaginal epithelial surface
is actually coated by a thin layer of fluid that includes endometrial, cer-
vical and vestibularsecretions, residues of urine and products of cellular
autolysis, and variable amounts of vaginal wall transudate [34,35].Addi-
tionally, the composition of the vaginal fluid varies according to age,
menstrual cycle and health status condition [31,36]. For instance, the
vaginal pH is acidic (3.5–4.5)in healthy women duringthe reproductive
age but it fluctuates along the different stagesof the menstrual cycleand
it is also dependent on coitus frequency, the amount of cervical mucus
present inthe vagina, the amount of vaginal transudate and italso varies
along the vagina being higher close to the cervix and lower at the ante-
rior fornix [2,35]. The maintenance of the pH is accomplished by lactic
acid bacteria, mainly Lactobacillus spp., since these microorganisms me-
tabolizeinto lactic acid themono and di-saccharides that result from the
autolytic breakage of desquamated vaginal cells glycogen [31].
The vaginal wall consists of various cell layers: nonkeratinized strat-
ified squamous epithelium, lamina propria, muscular layer and tunica
adventitia (covering only their proximal segments). The lamina propria
is constituted of connective tissue rich in blood and lymphatic vessels
draining to the internal iliac vein, this explaining why the absorbed
products do avoid the hepatic circulation as an initial passage [30].
The vaginal cell turnover is estimated to replace 10–15 layers in a
week [37]. The nonkeratinized stratified squamous epithelium, settled
on glycogen containing keratynocytes but also integrating other cell
types (such as macrophages and Langerhans' cells), is grounded on
the lamina propria consisting of fibroblasts, elastic and collagen fibers,
vessels and nerves,and defense cells, mainly polymorphonuclear leuko-
cytes and occasional lymph nodules [38]. The vaginal epithelial cells are
disposed according to different stages of differentiation, identifiable
through different keratins expression, such as K10 and K13, being the
differential expression arrangement function of the cell location within
the epithelium (Fig. 1)[39]. Numerous folds and microrridges called
“rugae”are present in the epithelium, largely increasing the vagina's
surface area and providing distensibility [7].
The vaginal innervation depends on two types of sources: a periph-
eral one providing a highly sensible lower quarter segment and an auto-
nomic fiber network in the upper vagina, which is more sensitive to
stretch than to touch or to painful stimuli. This explains why women
do not feel discomfort when using continuous intravaginal drug deliv-
ery systems [7].
Several conditions influence vaginal physiology: hormonal balance,
pregnancy, pH, microflora and age, being the last one the best biomark-
er for epithelium layer thickness, enzyme concentrations and vaginal
fluid production [41]. These vaginal characteristic changes influence
drug permeation as itdepends mainly on thesuperficial layer character-
istics, as thickness, cell tightness, and lipids composition and organiza-
tion in the intercellular space [25,42,43].
3. Drug absorption from the vagina
Drug dissolution in the vaginal fluid and epithelial penetration are
the two key steps for a drug to be absorbed through the vagina. As a re-
sult all factors associated with vaginal physiology and formulation pro-
file will greatly influence the success of drug delivery to the target [6,7].
Even though the vagina is not a real mucosa, drug transport is accom-
plished in a multi-way mechanism similar to the other biological
membranes. Drug absorption can occur passively or actively. Passive
mechanisms include the transcellular route, through the cells' mem-
brane and the paracelullar route, representing a diffusion process
through intercellular fluid and tight junctions [44,45]. Tight junctions
and other intracellular junctions (adherens junctions and desmossomes)
are present in the vaginal and cervical epithelium having a higher ex-
pression in the endocervix. Although the uppermost layers of the
ectocervical and vaginal epithelium are devoided of tight junctions,
these and other intercellular junctions have been identified right be-
neath the most apical epithelium layers. The study of these junctions
is particularly important to understand, for example, the invasion of mi-
crobes and drug permeation. Tight junctions are composed of trans-
membrane proteins (occludin, claudins and junctional adhesion
molecules —JAMs) whichcontact across the intercellular space and cre-
ate a seal to restrict paracellular diffusion of molecules across the epi-
thelial sheet. Furthermore, tight junctions have a structural role in
epithelial polarization by limiting the mobility of membrane-bound
molecules between the apical and basolateral domains of the plasma
membrane of each epithelial cell. In general, tight junctions are
15R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

responsible for sealing the epithelial barrier as well as the selective pas-
sage of small ions and fluid [44].
The active transporters generate gradients across the barriers, being
the mechanism either a primary transcellular process or a secondary
one. While the latter is indirectly coupled to adenosine triphosphate
(ATP) energy, the primary active transport directly utilizes ATP during
the transport cycle [46]. Nevertheless, previous studies on vaginal per-
meation of drugs show that most of the active substances permeate
the vagina through diffusion mechanisms [17]. Lipophilic substances,
such as steroids, are mainly absorbed by the transcellular route [31],
in contrast to hydrophilic drugs which follow the paracellular diffusion
mechanism [17]. Low molecular weight lipophilic drugs are more likely
to be better absorbed than larger molecules or even hydrophilic drugs,
independently of their molecular weight [5].
Several factors affect drug absorption from the vagina. Both vaginal
fluid amount and composition interfere in drug dissolution before
transport. While an excessive fluid content promotes a “washing”effect
that decreases drug retention, cervical mucus presence is able to in-
crease bioadhesion [31]. Estrogens, due to epithelial cell proliferation
stimulation, influence drug pharmacokinetics through the vagina, as a
consequence of increased blood irrigation, transudation and cervical
mucus production. Consequently, when a systemic action of a drug is
desirable, several factors shall be taken into account, likethe epithelium
layer thickness, the enzymatic and hormonal cyclic changes, the vaginal
pH (that interferes with ionization of electrolytic drugs) and sexual
stimulation and intercourse [47,48].
Additionally, vaginal administration of a substance, being either a
drug or an excipient, may cause irritation, inflammation and damage,
leading to barrier disruption and so enhancing not only its absorption
but also the penetration of infectious agents [25,49]. Drugs and excipi-
ents' physicochemical characteristics are also relevant for determining
their absorption [50,51]. The excipients present in the formulation
have also an important role both in drug release and absorption profiles
[7]. Molecules' lipophilicity, ionization, molecular weight, surface
charge and chemical nature represent the most relevant properties
that determine their ability to permeate both the apical and basolateral
membranes of the epithelial cells [24,52].
4. Vaginal drug permeation methodologies
The ethical and practical constraints to the use of humans and their
organs to assess drugs and formulations' efficacy and safety, led re-
searchers to create and develop alternative methods and models to pre-
dict drug permeation through the vagina. These include cells, tissues
(both organs and culture systems) and full animals [25,53–56]. Techni-
cal complexity, economic expenditures and ethical issues determine if a
model will either be valid, applicable and reproducible or if it is not
trustworthy to be used in laboratory research. Table 1 gathers some of
the main advantages and disadvantages of models to be applied for
predicting vaginal drug permeation.
4.1. In vitro models
Several in vitro studies have been conducted to characterize the vag-
inal barrier concerning drug permeation [57–60]. These models com-
prise cell-based and reconstructed tissue models.
Over the last decades, cell-based in vitro models for drug permeation
prediction allowed the use of simple, economic, reproducible and ethi-
cally accepted methodologies in the earlier stages of drug development.
These models use both immortalized cells and primary cell cultures that
are able to proliferate in monolayers, simulating the intended epitheli-
um, both structurally and functionally [24]. Cell culture models can be
easily manipulated, in terms of culture conditions and parameters, and
represent good drugabsorption predictors. Human cell lines or primary
cell cultures circumvent problems related to the use of animal tissue on
in vitro models [61].
The main limitation of in vitro models, result from the impossibility
of integrating cell external factors (diseases, age, hepatic and renal
Fig. 1. Schematic representation of nonkeratinized stratified squamous vaginal tissue.
Adapted from: Schnell, Citologia y Microbiologia de la vagina [40].
16 R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

functions, environmental characteristics) that are imperative for the or-
ganism function. Although, extrapolation of in vitro results to humans
must be done very carefully [24],thein vitro–in vivo correlation repre-
sents an important tool for prediction of in vivo pharmacokinetics.
Clark et al. in 2011 tried to compare results obtained in vitro with
in vivo for a preclinical evaluation of a microbicide in the form of a mi-
cronized and non-micronized drug. However, in vivo pharmacokinetic
results (in rabbits) failed to corroborate the higher permeability for
the micronized drug obtained with reconstructed tissue in vitro studies
[24].
Drug physicochemical and biopharmaceutical properties as well as
physiological and environmental organism characteristics should be
considered. Among them are drug solubility, acid dissociation constant
(pKa), drug permeation, octanol-water partition coefficient (logP) and
the environment fluid pH [62].Therefore,in vitro permeability studies
also play a key role in determininga formulation strategy in order to as-
sist solubility, dissolution and stability [63].
In the 90's Gorodeski et al. developed a method based on the har-
vesting of cervical–vaginal cells collected from women, aged from 22
to 49 years, undergoing hysterectomy. Initially, a primary cell culture
of human ectocervical epithelial cells was obtained (hECE) [64], that in-
cluded histologically normal cells only. This technique was applied to
permeation mechanistic studies. To this purpose, the cells were grown
on collagen-coated ceramic-based filters and differentiated into a
5–12 cell layers to form a squamous stratified epithelium, mimicking
the biological characteristic human cervical–vaginal epithelium [65,
66]. When these cells were compared to a cervical cell line (CaSki),
the latter ones showed to be simpler to maintain and a better alternative
to the primary cultures obtained from hysterectomy specimens. In this
study, it was also evidenced the epithelial nature of CaSki cells and
their improved differentiation when grown on filter support [67]. Al-
though they are easier to grow, maintain and test, cell line cultures cor-
respond to immortalized cells and consequently may not mimic real cell
behavior. From thispoint of view, primary cell cultures should be valued
since they represent a more reliable biologic model.
When different cellular models were developed and compared,
hECE, the primary culture, was settled as a model for ectocervical epi-
thelium, while ECE16-1 and CaSki, both immortalised cell lines,
expressed phenotypic characteristics of squamous metaplastic cervical
epithelium and endocervical epithelium respectively [68]. Later on,
CaSki endocervical cell line was shown to be able to form cell mono-
layers and even bi/trilayers, depending on cell seeding density, and
was proven to be useful for transport mechanisms studies [69–71].
These experiments resulted in interesting findings about the impor-
tance of tight junctions and their influence on transepithelial electri-
cal resistance (TEER) [66] and the increase of TEER by seminal fluid
[70]. They also contributed to improve the knowledge about the
modulation of vaginal permeability to pyranine (used as a model
drug for paracellular transport) by factors such as estrogen stimula-
tion and aging (due to changes in the resistance of both the lateral in-
tercellular space and the intercellular tight junctions) [72–76],or
extracellular ATP and Ca
2+
(due to their effects at the tight junctions
level) [67,75]. All this research resulted in substantial basic knowl-
edge for vaginal permeability studies. Table 2 resumes the in vitro
models developed for the study of drug permeation through the vag-
inal barrier.
Table 2
In vitro models developed for predicting drug permeation across the vaginal barrier.
In vitro model Source Permeation features studied References
Cell-based HEC-1A —cell line Human endometrial carcinoma. Drug permeation and solubility. Excipients influence on
drug permeation and solubility.
Transepithelial electric resistance.
[23]
hECE —human ectocervical epithelial
cells —primary culture
Human biopsy, histologically
normal cells.
Transepithelial electrical conductance.
Permeability to pyranine.
[64–66,68–71,75,84]
CaSki —human cervical epithelium
cells —cell line
Human epidermoid carcinoma. Transepithelial electrical conductance. [67,69]
C-33A —cervix cancer cell line cells Human cervical retinoblastoma. Transepithelial electrical resistance. [85,87]
Primary vaginal epithelial cell culture Human biopsy, healthy woman. Drug permeation. [78]
Reconstructed
tissue
Human reconstructed vaginal tissue
integrating normal or SiHa cell line
and Langerhans Cells (LCs)
Human biopsy, healthy woman.
Cell line from a squamous cell
cervical carcinoma.
Study of the transepithelial route followed by pathogens.
Safety evaluation of compounds applied on vaginal
epithelium.
[86–89]
VEC —vaginal–ectocervical tissue
model (EpiVaginal®)
Normal, human-derived vaginal
ectocervical epithelial and
dendritic cells —MatTek.
Transepithelial electrical resistance.
Sodium fluorescein leakage.
Inflammatory cytokine release.
Potential use for screening and assessment of the
irritation, penetration metabolism, or efficacy of active
ingredients or final formulations for vaginal application.
[56,79–83]
HVE® —Human vaginal epithelium A431 cells derived from a vulval
epidermoid carcinoma grown on a
polycarbonate filter —SkinEthic.
Screening and assessment of the irritation, penetration,
metabolism, or efficacy of active ingredients or final
formulations for vaginal application.
[89–91]
Table 1
Advantages and disadvantages of models applied for predicting vaginal drug permeation. Adapted from Costin et al. (2011) [38].
In vitro Ex vivo In vivo
Cell culture models
Reconstructed tissue
Tissue explants Laboratory animals
Advantages Cultures are relatively inexpensive and easy to grow. Full cell structure (epithelial,
connective, immune).
Better tolerance to formulations.
Parallel efficacy testing.
Complete organism features addressed: structurally,
functionally and physiologically.
Disadvantages No barrier function for cell cultures and no systemic
component.
Organotypic cultures tend to be more permeable than in vivo
and have no vascular component.
Limited number.
Institutional review board
approval required.
More technically demanding.
Variability.
Time and cost expensive.
Data still difficult to correlate to humans.
Regulatory and ethical issues.
17R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

Permeability studies using either primary cultures or cell lines of
vaginal/cervical epithelial cells have been described in the literature
and are herein discussed [72,74,76–78]. Cell lines represent more stan-
dardized models than primary cultures, since they are ready to use
without rising important inter laboratory variability results and inter-
pretations. Comparative studies on permeability differences between
primary cell cultures and cell lines represent an important topic to be
explored, for example using the TEER measurement. The simplicity of
these in vitro models can lead to limitations in terms of in vitro–in vivo
data correlation when compared to methods based on human vaginal
and cervical tissue explants. The importance of establishing these corre-
lations justifies the need to proceed with tests that better mimetize
in vivo mechanisms by using reconstructed tissue, tissue models
(ex vivo)andanimals(in vivo).
Regarding tests using reconstructed tissue, the Epivaginal® model
(MatTek Corporation, Ashland, United States of America), is a commer-
cial vaginal–ectocervical (VEC) tissue-like model widely used for
microbicides testing. It consists of a 3D-culture of non-transformed
human vaginal–ectocervical epithelial cells grown on polycarbonate
cell culture tissue inserts. The VEC tissue model has proven to be highly
reproducible and represents a non-animal method to assess the irrita-
tion of contraceptives, microbicides, and vaginal-care products [57,79].
Despite being largely used for studying prevention of pathogen
transmission [80,81] and tissue toxicity [79], the complexity of the
multi-layer structure makes it a promising option for permeability stud-
ies. Several other authors have already used EpiVaginal® as model to
predict vaginal permeability to chemicals [82,83] (Table 2).
4.2. Ex vivo models
Ex vivo models have been used for predicting drug permeation
through the vaginal barrier by using either human or animal vaginal tis-
sue. Ex vivo tissues allow not only for drug permeation assays but also
enables histological analysis to assess differences before and after drug
application/permeation [92]. While some authors may classify these
ex vivo experiments as in vitro experiments, the two concepts are con-
sidered to be different for the purpose of this review. Laboratorial recon-
structed tissues are herein classified as in vitro assays, as they are grown
in artificial milieu, while ex vivo assays require tissue excision from an-
imals or humans, and their use in conditions that preserve their original
biological and physical characteristics for a certain period.
Fresh human cervical explants are clinically obtained from women
undergoing planned hysterectomies. In the USA some repositories pro-
vide fresh samples for research institutes and industries, nevertheless it
is doubtful that the samples available are sufficient to extensive exper-
iments, such as the toxicological ones [38]. Researchers are concerned
Fig. 2. Comparison between H&E-stained vaginal epitheliumof: human (a), rabbit (b), rhesus monkey (c), pig (d), mouse (e), EpiVaginal from MatTek Corporation (Ashland, MA, USA)
(f) and HVE —Human vaginal Epithelium from SkinEthic (Nice, France) (g).
Reproduced from Reference [38] with kind permission from FRAME.
18 R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

Table 3
Permeability methods for predicting drug permeation across the vaginal barrier.
Membrane Membrane source and
preparation
Drug/substance Permeation experiment
characteristics
Quantification
method
References
Franz cell systems EpiVaginal®
VEC-100-FT
Commercially available. UC781, atenolol and
antipyrine
Receptor solution: 0.5 mL of the assay medium on
the basolateral side of the insert.
Sample collection: 200 μL were taken from the
receptor compartment at different times, and the
same volume of the fresh assay medium was
replaced back to the same compartment.
LC [83]
Porcine vaginal tissue Tissues collected from female laboratory pigs.
The tissue was frozen in SVF at −20 °C
immediately after sacrifice. Samples were cut in
1 cm square pieces and defrosted before
experiments at ambient temperature in the
presence of SVF.
Clotrimazole Receptor solution: 11 mL of phosphate-buffered
saline, at 37 °C.
Franz cells: area of 1.0 ± 0.1 cm
2
.
Sample collection:100 μLatfixed time intervals up
to 8 h.
Sample preparation:filtration.
HPLC [108]
CaSki cell monolayers Seeding and culture was performed on
transwell permeable supports for 8 days.
Polymeric nanoparticles of
dapivirine
TEER was measured to check monolayer formation
and integrity.
Receptor solution: HBSS with poloxamer 407, pH
7.3.
Sample collection: 500 μL samples were periodically
collected from the receptor chamber and the
volume replaced with fresh HBSS. Cell monolayers
were also collected either at 1 or 4 h after being
washed with PBS, lysed and analyzed for soluble
dapivirine.
HPLC [116]
Porcine vaginal tissue Obtained from a slaughter house, transported in
RPMI-1640 medium, and used within 2 h from
sacrifice. Thickness of 800 ± 300 μm.
Receptor solution: 8 mL of HBSS containing 0.2%
(w/v) of poloxamer 407 under magnetic stirring
(300 rpm).
Franz cells: exposure surface was 1.77 cm
2
.
Sample collection: samples were periodically
collected from the receptor chamber and the
volume replaced with fresh HBSS.
Fluorescence
microscopy HPLC
Bovine vaginal tissue Obtained from a local slaughterhouse and
transported in e isotonic NaCl solution.
Unstripped vaginal tissue was cut into 5 cm
2
segments.
Ketoconazole, propranolol,
furosemide, atenolol,
ranitidine, curcumin
Receptor solution: 7.5 mL, oxygenated Ringer buffer
at pH = 7.4, at 32 °C.
Sample collection: every 30 min up to 4 h.
Sample preparation: aliquots and extracted tissues
(cryotomized at −25 °C to 20 μm slices) were
prepared for liquid chromatography.
LC [103]
Cervical and vaginal
human tissue
Tissues collected from women undergoing
hysterectomy for benign conditions.
Tritiated water Receptor solution: 4.9 mL of phosphate-buffered
saline (PBS), pH = 7.4, at 37 °C.
Franz cells: 7 mm of diameter; 0.385 cm
2
of area.
Sample collection: 200 mL were removed from the
receiver compartment at various time intervals for
a total period of 2 h.
Sample preparation: Liquid scintillation cocktail,
2.5.mL.
Liquid scintillation [17]
(continued on next page)
19R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

Table 3 (continued)
Membrane Membrane source and
preparation
Drug/substance Permeation experiment
characteristics
Quantification
method
References
Human vaginal tissue Excised vaginal epithelial tissue from routine
reparative surgical procedures. Tissue was flash
frozen immediately in a liquid nitrogen
atmosphere and stored at −70 °C. Before
experiments as thawed slowly at 4 °C and cut
into cylinders, approximately 6 mm in diameter
and 4 mm deep, using a dermatological punch.
5-aminolevulinic acid Receptor solution:10 mL of PBS pH 7.4
Diffusion cells: modified Franz diffusion cell and
exposed to a water-soluble, ALA-loaded,
Sample preparation: the tissue was cryostatically
sectioned and the stratal concentration of
radiolabeled ALA determined using scintillation
spectroscopy.
Scintillation
spectroscopy
[106]
Ussing Chambers Porcine vaginal tissue Obtained from a slaughter house.
Tissues were thawed in PBS (pH 7.4) for 10 min.
The thawed specimens were cut into 4 mm
diameter sections.
17 β-estradiol, r-arecoline,
vasopressin, oxytocin and
water
Receptor solution: PBS pH = 7.4, at 20 °C
Diffusion cells:flow-through diffusion cells
(exposed areas 0.039 cm
2
)
Sample collection: PBS at 20 °C was pumped
through the acceptor chambers at a rate of 1.5 mL/h
and collected, by means of a fraction collector, at
2 h intervals for 24 h (100 μm).
Sample preparation: to each sample collected, 15 ml
scintillation cocktail was added.
Liquid scintillation [104]
Millipore, Durapore®
0.45-μm pore size
Commercially available. miniCD4 M48U1 Receptor solution: 13 mL of SFV without BSA,
at 37 °C.
Diffusion cells: area of 2 cm
2
.
Sample collection: 1 mL at fixed time intervals up to
24 h.
Surface plasmon
resonance
[117]
Vaginal macaque
tissue
Collected from naïve female Cynomolgus
macaques (Macaca fascicularis) and rinsed with
Ringer solution (pH 6.8).
M48U1 Receptor solution: Ringer solution
Sample collection: until 2 h, 0.1 mL was removed
Sample preparation: extraction using ultrasounds
Flow-through
diffusion cells
Porcine vaginal tissue Obtained from a slaughter house.
Tissues were cut into disks of 8–10 mm
diameter.
Tritriated water Receptor solution: PBS 0.01 M, pH7.4 at 37 °C,
pumped through the receptor chamber at a rate of
1.8 mL/h
Diffusion cells: 5 mm diameter orifice, with an
exposed epithelial surface of 0.20 cm
2
.
Sample collection: the perfusate was collected in
scintillation vials at 60 minute intervals for up to
19 h using a fraction collector.
Sample preparation: the samples were mixed with a
scintillation cocktail.
Liquid scintillation [118]
Human vaginal tissue Obtained from excess tissue removed from
postmenopausal patients after vaginal
hysterectomies. Only normal specimens were
used.
Tritium radioisotopes of water,
17β-estradiol, arecoline,
arecaidine,
Sumatriptan, cyclosporine,
benzalkonium chloride,
benzo[a]pyrene, vasopressin,
4.4-kd and 12-kd
fluorescein-isothiocyanate-
labeled dextrans.
Receptor solution: PBS pH = 7.4, at 20 °C
Diffusion cells: area of 0.039 cm
2
,flow-through
chambers.
Sample collection: PBS at 20 °C was pumped
through the acceptor chambers at a rate of 1.5 ml/h
and collected, by means of a fraction collector, at
2-h intervals for 24 h (100 μm).
Sample preparation: to each sample collected, 15 ml
scintillation cocktail was added.
Liquid scintillation [76,77,94–97,
119–126]
20 R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

with the difficulty to obtain and maintain human explant specimens at
the laboratory. To overcome these difficulties, freezing of specimens has
been considered and no negative influences in permeability parameters
were foundfor this tissue conservationmethod. In fact, no statistical dif-
ferences were found between fresh and frozen tissue permeability re-
sults when tissues were collected 1 h after the surgical excision,
transported in PBS or culture media at 5 °C, frozen with methanol/dry
ice or liquid nitrogen, and kept frozen at −80 °C [17]. Previous studies
conducted by van der Bijl et al. showed that freezing at −85 °C had
no effect on tritiated water permeation through vaginal tissue, after tis-
sues transport in a transport fluid, and transferred to laboratory within
1 h, snap-frozen in liquid nitrogen and stored at −85 °C for up to
6 months [93–97]. Although, permeability studies may not be influ-
enced by tissue freezing–thawing, the impact on vaginal irritation is
not clear, since cellular viability might be affected [38,98].Meanwhile,
the difficulties encountered with the human tissue samples impelled
the demand for other alternatives, such as animal tissues.
Vaginal tissue obtained from different animal species such as ro-
dents, rabbits, monkeys, cows and sheep have been used for permeabil-
ity studies [99–103]. Vaginal absorption may be significantly different
when comparing the animal and the human models.
Bovine vaginal tissue was used to test drugs permeation, as surro-
gate for human tissue due to anatomical and physiological similarities
[103]. Nonetheless, bovine epithelium exhibits significant histological
differences when compared with human vaginal epithelium and is not
widely applied. In contrast, porcine tissue represents the animal
model most commonly applied for this purpose. Porcine ex vivo tissue
specimens are convenient, since they are simple to handle, inexpensive
to obtain and easy to work comparing to the whole animal. Human and
porcine vaginal tissues present substantial histological similarities [25,
98] (stratified squamous epithelium supported by connective tissue –
Fig. 2). Additionally, porcine vagina can be easily accessed through
local slaughterhouses.
Although, generally, porcine vaginal tissue seems a good ex vivo per-
meability model for human vaginal tissue extrapolation it isof great im-
portance to validate a permeability study concerning other tissues or
in vitro tests. It has already been shown that for hydrophilic molecules,
(water and vasopressin, for example), the porcine vaginal tissue is an
accurate in vitro permeability model of human tissue, however for
more lipophilic molecules (such as oxytocin) the flux through porcine
vaginal tissue was 53% higher than the corresponding estimated value
through the human vaginal tissue [104].Ex vivo tissues are mainly test-
ed for drug permeation usingone of three established techniques: Franz
cell systems, flow through diffusion cell and Ussing chambers.
4.3. Permeation tests
The United States Pharmacopeia (USP) clearly defines the perfor-
mance tests for topical drug products, including vaginal administration
products, which are focused on the assessment of in vitro drug release. It
is defined that this test must be performed in vertical diffusion cell
(VDC) systems, which by their simplicity can provide reliable and repro-
ducible measurements of drug release from semisolid formulations.
Franz cells represent the most often used VDC, on the top of which
200–400 mg of the testing formulation is placed, over the membrane
to be studied. The application site can be variable in size, but is typically
of 15 mm of diameter. For vaginal drug delivery systems the assay tem-
perature must be kept at 37 ± 1 °C. Samples of the recipient fluid are
collected up to 4–5 h, and the volume withdrawn is replaced with
fresh medium. Sink conditions must be assured, meaning that the re-
ceptor medium must have a high capacity to dissolve the drug, and
the receptor media should not exceed 10% of the concentration of the
standard at the end of the test. This test should be done with two runs
of 6 cells in order to document the release rate [105].
Although USP recommendations point towards the use of synthetic,
inert and highly permeable membranes in the context of these drug
release studies, the interest in performing in vitro permeability studies
using biological membranes is evident. Biological tissues/membranes
are more similar to the in vivo conditions and may provide information
on bioavailability of the drug. However, th ey are also more complex, and
data obtained can be more difficult to interpret and discuss. Generally,
the difficulties to be encountered are related to tissue preparation and
system setting-up. The membrane preparation depends on tissue pro-
venience. Since human tissues are difficult to obtain, frozen and thawed
samples are frequently used since previous studies demonstrated that
this procedure does not affect tissue permeability [96,106].
Permeation systems intended to study drug release from vaginal
dosage forms include Franz cell systems, flow through diffusion cells
and Ussing chambers, further described in Sections 4.3.1, 4.3.2 and
4.3.3, respectively.
Perfusion cells are commercially available in different configurations
and sizes concerning tissue exposure areas, donor and receptor cham-
bers. In terms of exposed tissue, the surface areas are quite variable
ranging from 0.039 to 3.14 cm
2
in published studies (see Table 3).
Also the receptor chamber volumes are diverse. Every detail must be
addressed in experiments preparation, since they should not only fol-
low pharmacopoeial recommendations, but also, integrate physico-
chemical characteristics of the substances under study. In addition to
maintaining a physiological temperature throughout the study, the se-
lection receptor fluids should be adequate since it is crucial to preserve
tissue integrity over time, in order to mimic physiological conditions.
The temperature should also be adequate and identical to in vivo
conditions.
The Organisation for Economic Co-operation and Development
(OECD) is an intergovernmental organization which harmonizes poli-
cies in developed countries and has issued several publications
concerning the safety assessment of chemicals and chemical prepara-
tions (www.oecd.org). Since, for example, vaginal lubricants can be
classified, for marketing purposes, as medical devices or even hygiene
products (i.e. they are not medicines, and so do not necessarily follow
the pharmacopeia specifications), manufactures should follow the
same methodology on characterizing products to standardize proce-
dures. Patterning the manufacture and testing methods provide better
awareness of the product, even on already commercialized products
[107]. The OECD series on Testing and Assessment Number 28, clearly
describe for skin permeability testing, the types of studies (in vivo or
in vitro) and the detailed procedure data. These include the species to
be selected together with the number, sex, housing and feeding condi-
tions of animals used in in vivo studies; and for in vitro studies the diffu-
sion cells type, the receptors fluids characteristics and skin preparation.
Additional considerations regarding the testing substance are also de-
scribed. No specific guidelines are available for vaginal (or genital)
products. Since the vaginal route plays an important role both on local
and systemic delivery, the availability of validated vaginal models and
regulatory recommendations similar to those for skin permeability
studies is of extreme relevance. Also, the European Union Reference
Laboratory for alternatives to animal testing (EURL-ECVAM) has not
yet validated in vitro methods for vaginal route studies, as it has been
done for skin irritation and corrosion (OECD test guidelines 431 and
439). Nevertheless, since there are commercially available in vitro re-
constructed tissues mimicking the vaginal epithelium (such as
Epivaginal® and HVE®), they might need, in a future, an international
recognition (meaning validation), so they can be widely applied with
reinforced confidence on the results obtained.
4.3.1. Franz cells
Several in vitro studies have already been conducted to characterize
the vaginal barrierin terms of drug permeation using a Franz cell system
[57,58]. Additionally this model constitutes the methodology of refer-
ence in the USP for topical drug products. The system is composed of
two compartments: a donor compartment (upper chamber) and a re-
ceiver compartment (lower chamber) (Fig. 3). A membrane is placed
21R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

between the two compartments. The formulation to be tested is placed
on the top of the membrane, in the donor compartment which does not
need to be filled with a liquid. Heated water circulation is maintained
around the receptor chamber all over the process, usually at 37 °C to
mimic human body temperature. The stirbar guarantees the receptor
solution homogeneity. Samples can be accessed over time by collecting
aliquots through the sampling port.
A phosphate-buffered saline (PBS) solution, with a physiological pH
is usually applied in the receptor chamber [17,108] despite oxygenated
Ringer buffer has also been used [103]. Tissue thickness and area must
be taken into consideration in results calculations. USP recommends
the use of synthetic and inert membranes in which the product to test
is placed, letting the system to saturate for 30 min (equilibration peri-
od). The USP indicates that the epithelial side of tissues must face the
donor solution. Aliquots from the receptor compartment are collected
at predefined time intervals. The aliquots volume withdrawn must be
replaced with the same volume of receptor solution to ensure sink con-
ditions. Afterwards, aliquots s hould be analyzed through div erse analyt-
ical techniques for identification of drug in the collected sample.
Membrane/tissue should also be checked for histological modifications
after the permeability study.
Steady-state flux (J
ss
) and apparent permeability coeffient (P
app
)can
be calculated based on the Fick's First Law of Diffusion, with the follow-
ing equations —Eqs. (1) and (2).
Jss ¼Q
At ð1Þ
Papp ¼dQ
dt A Cd

60 ð2Þ
being Qthe quantity of substance crossing membrane [counts per min-
ute (cpm)],Athe area of the membrane exposed (cm
2
), and tthe time of
exposure (min); the units for J
ss
are cpm cm
−2
min
−1
. In the second
equation, dQ/dtis the slope of the curve of Qversus t,Aremains the sur-
face area and C
d
is the initial concentration of the compound in the
donor compartment (cpm/cm
3
). Therefore, the units for P
app
are cm/s
[93].
The permeability chamber design may be responsible for results var-
iability. Classic Franz cell system used in permeation studies can lead to
stasis and/or accumulation of perfusate in the receptor chamber, which
can be overcome by using a diffusion flow-through system [58].
Concerning the experimental conditions, temperature stands as an es-
sential parameter to be controlled since drug diffusion depends on tem-
perature. Also, tissue origin and the experiment duration must be
optimized to get the best profit of permeability studies [25]. Additional-
ly, researchers should be aware of intrinsic biological variability which is
obviously more pronounced in tissues and reconstructedtissues, than in
synthetic membranes.
Several investigation studies have already been performed using
Franz cell systems to perform in vitro drug release studies on vaginal
products (Table 3).
4.3.2. Flow through diffusion cells
Flow-through diffusion cells have been developed to overcome an
identified limitation of static diffusion chambers: the possible decrease
of diffusion gradient following the accumulation of the permeating
drug in the receptor compartment. These diffusion cells present the
same principle as those previously described since they are based on
the product application at one side of the tissue/membrane (donor),
with permeation monitored, through aliquot collection and analysis,
on the receptor side. However, these systems are characterized by the
use of a perfusion fluid, bellow the membrane surface, to collect the per-
meating substance and allow for a concentration gradient across the tis-
sue that better resembles the in vivo conditions. Also, the sampling can
be further facilitated by collecting the effluent using a fraction collector
(Fig. 4). The flow-through diffusion cell allows for diverse benefits: au-
tomatic sampling; easy maintenance of sink conditions; mimicking the
physiological assessment of percutaneous absorption; simulation of the
blood flow subjacent to biological membranes [109].
However, the receptor volume is critical since, to completely wash
the permeating substance, the pumped volume must be many times
the volume of the receptor. This requires the receptor volume to be
small so that the volume of effluent from the cell is manageable [110].
A different type of modified flow diffusion cells was proposed by
Bonferoni et al., characterized by a flow stream on the donor chamber
in order to measure the washability of semi-solid formulations [111].
4.3.3. Ussing chambers
Ussing chambers are also used for the experimental measure of drug
permeation through biological barriers. These systems allow not only
for temperature control, but also for oxygen circulation in the two
chambers and measurements of electric parameters to monitor tissue
viability [112].
The Ussing chamber system, introduced by Hans Ussing in the early
1950s [113], is composed of a chamber, a water jacket to maintain phys-
iological temperature, a gas stream system to maintain a physiological
Fig. 3. Schematic representation of a Franz cell. (A) donor compartment; (B) membrane;
(C) sampling port; (D) stirbar; (E) water outlet; (F) water inlet.
Fig. 4. Schematic representation of a flow through diffusion chamber. (A) donor compartment; (B) membrane; (C) receptor compartment.
22 R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

buffer while providing a gas lift circulation and, if needed, an amplifier
and/or a data acquisition system (Fig. 5). Although they were initially
developed to study ion transport mechanisms across the epithelia,
Ussing chambers have also been applied to drug permeation studies
(Table 3). Epithelia are polar structures possessing an apical (also re-
ferred to as mucosal) and basolateral (or serosal) side. The electrolytes,
non-electrolytes, and H
2
O movement across the membrane once quan-
tifiable can be extremely useful in substance permeation studies. Ussing
chambers not only support native human or animal tissues but also
membranes derived from cell monolayers and reconstructed tissues.
Specifically, cells can be grown on monolayers in a culture insert and
then placed on a Snapwell chamber, which is provided by the apparatus
manufacturer and can be used in the Ussing chambers system. In the
Snapwell system the drug to study is dissolved in the donor fluid. For
formulation studies several difficulties may arise as is the case of
semi-solids testing since the viscosity of the product or the solution ob-
tained after dissolution may impair gaseous and nutrient exchange
therefore negatively impacting cell viability and the obtained results.
Semi-solid formulations can be directly applied on tissues or for in-
stance on reconstructed tissues while for cellular permeation studies,
a previous dilution is necessary to avoid the occlusive effect.
The barrier to permeate is placed between the two chamber halves
(Fig. 5). Diverse technical variations of these systems have been devel-
oped to meet the appropriateness for different experiments including
vertical and horizontal Ussing chambers. The central purposes of Ussing
chambers technique are electrophysiological and diffusion-based stud-
ies, or moreover a combination of both [114].
The Ussing chamber system consists of two functional parts: the
chamber itself and the electrical circuit. The electronic circuit measures
the current (I)andvoltage(V) which allow for the calculation of resis-
tance (R), and also more complex parameters like impedance and ca-
pacitance [115]. Depending on the type of fluid circulation Ussing
systems can be classified into two types: the circulating chamber and
the continuously perfused chamber. The circulating chamber is U-
shaped, embraces the experimental solution and can be heated and
gassed (air, CO
2
,O
2
or N
2
), in order to oxygenate the liquid contents
and guarantee complete convection of the liquid. The U-shape ensures
equal hydrostatic pressure on both chamber halves. This chamber is
considered to be robustand simple to use. Continuously perfused cham-
bers are not commercially available, but can be manufactured by design
with specificconfigurations. The main purpose of these chambers is to
minimize the hydrostatic pressure, avoiding damage to the testing tis-
sue. The flow of the perfusion solutions is accomplished by using peri-
staltic pumps. The immerse solutions to be applied in the chamber are
automatically delivered from reservoirs mounted above the chamber.
The flow rate of injection can be regulated. The majority of the chambers
are made of Teflon® or Lucite® and are available at various sizes and
shapes [114].
Epithelium is distinct from other tissues due to its polarity and tight-
ness. This characteristic derives from the asymmetric distribution of
proteins to either the apical or the basolateral side of the cellular mem-
brane. The tight junctions seal adjacent cells, and its formation and per-
meability determines the resistance (R) and, consequently tissue
integrity. The resistance is calculated through the following equation —
Eq. (3).
R¼ρl
Að3Þ
where ρis the specific resistance module of the material, lthe length or
thickness of the material (which should be constant for each tissue
preparation), and Athe surface area. Considering a specific tissue, R
can be seen as a sum of resistors, being R
a
the resistance of the apical
membrane and R
b
the resistance of the basolateral membrane, shunted
by a parallel resistor R
shunt.
Therefore, the total transepithelial resistance
R
t
is defined by the Kirchhoff's law expressed in Eq. (4).
Rt¼RaþRb
ðÞRshunt
RaþRbþRshunt
ð4Þ
R
t
reflects directly the tissue integrity and can be easily calculated
using the Ohm's law —Eq. (5).
Rt¼ΔV
ΔIð5Þ
The easier way to assess R
t
is to apply a voltage and measure the
resulting change in current (called “voltage clamping”). In addition,
the ion transport across epithelial tissues generates a transepithelial
voltage, V
te
. Another electric parameter to take into account isshort cir-
cuit current (I
sc
). This current is defined as the charge flow per time
when the tissue is short-circuited and is measured when V
te
is clamped
to 0 mV. The amount of current required is adjusted andregistered. The
values different to 0 mV enable a estimation of R
te
, a value that can be
applied in the following equation to calculate I
sc
—Eq. (6).
Isc ¼Vte
Rte
ð6Þ
In summary, an experiment using Ussing chambers shall consist on
three steps. A preparation phase must be performed to ensure cham-
bers, solutions, electrodesvalidity and readiness. At this time the cham-
bers must be mounted without tissue for watertight verification of the
system, while temperature and gassing can also be tested. As a second
step it is recommended to perform a control experiment to check for
electrical interferences (noise) and offset voltages. This also allows for
resistance estimation of the empty chambers. Only after all these verifi-
cations,the tissue experimentshould be accomplished (third step). The
tissue is mounted between the half chambers, allowed for an
Fig. 5. Schematic representation of a circulating Ussing chamber vertical system, Navicyte
™. (A) sampling port; (B) current electrods; (C) voltage electrods; (D) gasification tube;
(E) membrane.
23R.M. Machado et al. / Advanced Drug Delivery Reviews 92 (2015) 14–26

equilibration period, and after a baseline is reached the experiment can
begin [115].
Table 3 summarizes the permeability studies (either based on Franz
Cell systems, Ussing Chambers and flow through diffusion cells) under-
taken for vaginal tissue by several investigation groups.
4.4. In vivo models
In vivo models represent the most complete approach to achieve ex-
perimental data. However, they are associated with ethical issues, re-
quire high cost expenditures and are time consuming. Few animals
have been studied for in vivo vaginal formulation testing, specifically
for vaginal irritation [127–129]. The rabbit vaginal irritation (RVI)
model is mostly used since it is the only approved model for the regula-
tory acceptance of new products [38].
Vaginal permeability testing is scarce on in vivo studies, although it
could be of great interest especially for systemic drug delivery systems,
provided that it is previously validated, ethic, safe and with benefitto
new drug and formulation development. Acartürk et al. used normal
and ovariectomized rabbits to mimetize different reproductive physio-
logical status, particularly post-menopausal human vagina. Explants of
vaginal tissue from these animals were further used for comparative
in vivo/ex vivo vaginal permeability and enzymatic activity studies
[130]. This model was also applied in the study of nonoxynol-9 as
spermicide [131].
More recently, the pig model has also been used for in vivo experi-
ments. Female large white pigs were used for the determination of zido-
vudine and polystyrene sulfonate in plasma and vaginal tissue, after the
application of an intravaginal bioadhesive polymeric device for up to
28 days. At the end of this period, vaginal tissue was collected for histo-
logical analysis, and for substances extraction. Both zidovudine and
polystyrene sulfonate were found in lowconcentrations in plasma, indi-
cating the high retention in the vagina. Additionally no histopathologi-
cal toxicity was evidenced for this intravaginal bioadhesive polymeric
device [132].
5. Conclusions
The vagina is a promising route for drug delivery intending both
local and systemic drug delivery. Over the last decades, efforts have
been made to conduct investigation to a level of high comprehension
of vaginal drug absorption mechanisms. The understanding and appli-
cation of several techniques to predict drug permeation through the
vaginal barrier contributes to the successful selection of drugs and ap-
propriate formulations in the early stages of development. Thus, it is
of great importance to implement accurate and reproducible methods
to predict drug permeation. The in vitro and ex vivo tests should be
privileged, once an optimal in vitro–in vivo correlation has been
established. Furthermore, tissue explants (ex vivo), being more repre-
sentative in structure and presenting more viability, shall be preferred
over in vitro cellular methodologies. Standardization and validation of
methodologies to beused by different research groups will beobviously
valuable for uniform interpretation and extrapolation of test results.
Moreover, these tests may be particularly important for the develop-
ment and characterization of new products intended to reach the mar-
ket since they provide vital information for marketing and regulatory
authorization purposes.
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