You are on page 1of 9

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Development of Smart Semisolid Formulations to Enhance Retinoic


Acid Topical Application
LUANA PERIOLI,1 CINZIA PAGANO,1 MORENA NOCCHETTI,1 LOREDANA LATTERINI2
1
2

Dipartimento di Scienze Farmaceutiche, Universit`a degli Studi di Perugia, Perugia 06123, Italy
Dipartimento di Chimica, Biologia e Biotecnologie, Universit`a degli Studi di Perugia, Perugia 06123, Italy

Received 11 June 2015; revised 15 July 2015; accepted 23 July 2015


Published online 17 August 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24612
ABSTRACT: Retinoids play a very important role in the topical treatment of acne vulgaris. However, their use is restricted because of
the limited photostability responsible for local adverse effects as erythema, dryness, itching, and stinging. In this way, the therapeutic
efficacy of such molecules is strongly reduced, resulting, at the same time, harmful for the patient upon light exposure. Thus, a suitable
technological strategy is necessary to increase retinoid stability in order to have a product both safe and efficacious. With this aim, new
inorganicorganic hybrids based on tretinoin (retinoic acid, RET) and hydrotalcite-like compounds (HTlc) have been prepared and well
characterized by X-ray powder diffraction, inductively coupled plasma spectrometry, thermal analyses, scanning electron microscopy, and
UVVis spectrophotometric measurements. Such hybrids, namely, ZnAl-HTlc-RET and MgAl-HTlc-RET, were formulated as simple gels
for topical use and submitted to further studies in order to evaluate their rheological properties, photostability, and RET release capability.
The RET photostability resulted improved upon intercalation into HTlc, both in MgAl-HTlc and ZnAl-HTlc, as proved by the data acquired
during irradiation of the sample at 366 nm. This strategy is suitable for the realization of safe, efficacious, and compliant topical formulations
C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:39043912, 2015
for acne treatment. 
Keywords: retinoic acid; hydrotalcite; inorganicorganic hybrids; rheology; usability; solidity; drug delivery; Gels; X-ray diffraction;
fluorescence spectroscopy clays

INTRODUCTION
Acne vulgaris, acne infantum, acne fulminans, and induced
acne, deriving from exposition to cosmetics, pharmaceutical
treatment with steroids or face autoinduced trauma, are the
main cutaneous chronic inflammations affecting the sebaceous
follicles.1 Acne vulgaris, the most frequent form of these diseases, is a multifactorial chronic dermatosis that appears generally during puberty as well as in adult age.
Acne has four main pathogenetic mechanisms: comedogenesis (sebaceous follicle obstruction), increased sebum production, Propionibacterium acne colonization, and inflammation.2
It is characterized by various clinical signs as seborrhoea,
comedones, erythematous papules and pustules, less frequently
nodules, deep pustules, or pseudocysts.3,4 Comedones, deriving
from an excessive keratinization in the sebaceous follicle, represent the starting point for the inflammation process beginning.
The hyperproliferation of keratinocytes is stimulated by the
androgenic hormone testosterone, able to bind specific membrane receptors of this kind of cells, and the conversion of such
hormone in dihydrotestosterone is particularly enhanced (from
two to 20 times more) in the acneic skin.5,6
The androgenic hormones provoke sebaceous gland hypertrophy responsible for the increased sebum production. A microbial flora, a stagnant cap of lipids and desquamed cells
are present in the follicular canal. These components represent
an optimal microenvironment for anaerobic bacteria growth,
Correspondence to: Luana Perioli (Telephone: +39-075-5855133; Fax: +39075-5855163; E-mail: luana.perioli@unipg.it)
This article contains supplementary material available from the authors upon
request or via the Internet at http://wileylibrary.com.
Journal of Pharmaceutical Sciences, Vol. 104, 39043912 (2015)

C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

3904

particularly Propionibacterium acnes, Propionibacterium


granulosum, Staphylococcus epidermidis, and Malassezia
furfur.1 The inflammation process is mainly because of lipases,
produced by Propionibacterium acnes, able to degrade triglycerides into free fatty acids acting as comedogenic agents because of their implication in the anomalous keratinization of
the infrafundibolar epithelium. This process provokes the obstruction of the follicular neck generating the irritation of this
area.
Acneic lesion treatment changes with relation to the inflammation process step. In mild forms, an accurate skin cleaning
is enough; acid skin cleaners must be preferred because of their
ability to reduce the sebaceous secretion and generate unfavorable environment for microbial growth.
Acne can be treated by using suitable cosmetic/
dermatological products for topical use having an appropriate
composition in terms of active ingredients and excipients. The
active ingredients generally show different properties as keratolitic and bacteriostatic activity (e.g., benzoyl peroxide), keratolitic, sebum regulatory and anti-inflammatory activity (e.g.,
azelaic acid), antibacterial (e.g., eritromicin), and epithelial cell
growth modulator (e.g., retinoids). It is very important to avoid
fat substances (e.g., lanoline, isopropilmiristate) as formulation
excipients because they are able to obstruct the pores resulting
comedogenic.7
Retinoids are natural and synthetic molecules, from which
originates Vitamin A, a liposoluble vitamin involved in the
pathways regulating vision, in the differentiation and proliferation of epithelial structures, in the carcinogenesis regulation
and in the immunitary response. Vitamin A skin application
is able to promote desquamation process by reducing the production of keratin and desmosomes. Among retinoids, retinoic
acid (RET) is the most used molecule for topical treatment

Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

of acne vulgaris. In this regard, it is important to underline


that two isomeric forms of RET are available: tretinoin (all
trans-RET) and isotretinoin (cis-RET). The two forms differ from the induced adverse reactions, minor in the case of
isotretinoin, resulting for this reason more tolerated.8
The main problem, associated to topical use of RET, is the
variable tolerability because of the molecule low stability to
light (such as light sensitivity) responsible for local adverse
effects as erythema, dryness, itching, and stinging.9
In order to have an effective and safe product for topical
treatment of acne vulgaris, it is important to develop a strategy
to increase RET photostability to avoid or reduce the abovedescribed problems.
With this objective, and on the basis of successful results
obtained for other cosmetic ingredients,1012 the present paper deals with the study of new inorganicorganic hybrids of
tretinoin intercalated in hydrotalcite-like compounds (HTlc).
These hybrids have been planned, prepared, and fully characterized. After this, suitable formulations have been developed
and studied with the aim to obtain a new product able to provide
reduced side effects, improved usability, and user compliance
in comparison to tretinoin topical products, currently available
on the market.

MATERIALS AND METHODS


Reagents
ZnCl2 (Brenntag), urea (ChemischeFabrikLehrte), RET
(Tretinoin-USP 29/EP5) (SOLMAG s.p.a. ARTHA), and propylene glycol furnished from Comifar Perugia.
AlCl3 6H2 O, MgCl2 6H2 O, K2 CO3 were purchased from Carlo
Erba (Pomezia, Italy). Hydroxyethyl cellulose (HEC; Euchem.
Morelli e Camassei) was purchased from Farvima Perugia
(Italy). Deionized water was obtained from reverse osmosis process with Milli Q System (Millipore, Rome, Italy). Other chemicals and solvents were of reagent grade and were used without
further purification.
R
) was purCommercially available isotretinoin gel (Isotrex
chased in pharmacy.
The fluid used in the release studies was a solution of K2 CO3
0.025 M and tetrahydrofuran (THF) in the ratio 70:30 (v/v).
HTlc Syntheses
Crystallized [Mg0.66 Al0.34 (OH)2 ](NO3 )0.34 0.4H2 O and [Zn0.70
Al0.30 (OH)2 ](NO3 )0.30 0.4H2 O were obtained by titration with
HNO3 0.1 M solution the corresponding carbonate forms dispersed in a 0.1 M NaNO3 solution (1 g/50 mL).13 Carbonate
anions are strongly held and difficult to exchange, whereas nitrate anions are more suitable for anionic exchange reactions14 ;
thus, carbonate form of HTlc was converted into nitrate form.
MgAl-HTlc and ZnAl-HTlc in carbonate form were obtained
by coprecipitation of bivalent and trivalent metal cations accomplished by the hydrolysis of urea.15 The synthetic hydrotalcites in carbonate form were obtained by adding solid urea
to a 0.5-M metal chloride solution. The Al(III)/(Al(III)+M(II))
and urea/(M(II)+Al(III)) molar ratio were 0.33 and 3.3, respectively. The hydrolysis of urea, inducing slow pH increase, led
to the precipitation of metals in well-crystallized HTlc carbonate form. The mixture was heated at 100C under stirring for
36 h. The final products (MgAl-HTlc-CO3 and ZnAl-HTlc-CO3 )
DOI 10.1002/jps.24612

3905

were recovered, washed with water to eliminate chlorides, and


stored in desiccator over P2 O5 at room temperature.
HTlc-RET Syntheses
The intercalation products MgAl-HTlc-RET and ZnAl-HTlcRET were prepared by one-pot THF-assisted grinding intercalation method as described by Conterosito et al.16 For both the
intercalation products, the molar ratio HTlc/RET 1:2 was used;
MgAl-HTlc and ZnAl-HTlc in nitrate form and RET (converted
in sodium salt form) were used as starting materials.
Inductively Coupled Plasma Spectrometry
Metal analyses were performed by Varian 700-ES series inductively coupled plasma-optical emission spectrometers (ICPOES) using solutions prepared by dissolving about 50 mg of
the sample in some drops of concentrated HNO3 , then diluted
to 250 mL with Milli Q water. A successive dilution, ratio
1:3 (v/v), was carried out.
X-Ray Powder Diffraction
The X-ray powder diffraction (XRPD) patterns were performed
with a diffractometer (PW 1710; Philips, Lelyweg, The Netherlands), using the Ni-filtered Cu K" radiation that works at
40 KV, 30 mA with goniometer PW 1820 and graphite
monochromator for diffracted ray. Diffractograms were registered with step scanning method (step size 22 = 0.03) and
were elaborated by PC-APD program.
Thermogravimetric Analysis
Coupled thermogravimetric and differential thermal analyses
were performed with a Netzsch STA 449C apparatus, in air
flow and heating rate of 10C/min to determine the weight loss
(water and drug) as a function of increasing temperature.
Differential Scanning Calorimetry
Differential scanning calorimetry analyses were performed using an automatic thermal analyzer (Mettler Toledo DSC821e)
and indium standard for temperature calibrations. Holed aluminum pans were employed in the experiments for all samples,
and an empty pan, prepared in the same way, was used as a
reference. Samples of 36 mg were weighted directly into the
aluminum pans and the thermal analyses of samples were conducted, at a heating rate of 5C/min, from 25C to 200C.
HyperChem
HyperChem program (HyperchemTM , 2000) has been used
to obtain the computer intercalation compound simulated
model.17
Scanning Electron Micrographs
Scanning electron micrographs (SEM) analyses were taken
with a Philips SEM 501 (PW 6703). Before observation, the
dried samples were sputtered and coated, for approximately
5 min under an argon atmosphere, with gold-palladium.
RET Release Capability from HTlc-RET Intercalation Products
The experiment was performed using aqueous solution of
K2 CO3 0.025 M18 mixed to THF (70:30, v/v) as dissolution
medium. The intercalation products ZnAl-HTlc-RET (220 mg)
and MgAl-HTlc-RET (170 mg), containing an amount of drug
Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

3906

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 1. Compositions of Gels Containing Free RET and Intercalated


in MgAl-HTlc and ZnAl-HTlc
RETH Gel

MgAl-RET Gel

ZnAl-RET Gel

RET

0.05 g

Ethanol 96%
Propylene glycol
HEC
Deionized water

20 g
25 g
5g
49.95 g

0.084 g
Corresponding
to 0.05 g of RET
(loading 58.9%)
20 g
25 g
5g
49.91 g

0.110 g
Corresponding
to 0.05 g of RET
(loading 45.5%)
20 g
25 g
5g
49.89 g

between the excitation and the emission light. The spectra were
corrected for the response of instrument components at each
wavelength. The traces of fluorescence intensity as function of
irradiation time, which gave information on the photochemical
stability of the samples, were recorded fixing the excitation and
emission wavelengths and monitoring the emission intensity
during the irradiation. Steady-state irradiation experiments
were performed using a xenon lamp as light source, selecting
the excitation wavelength (366 nm) by a monochromator and
setting a band-pass slit of 35 nm.

RESULTS AND DISCUSSIONS


corresponding to 100 mg of free RET, were placed in two different closed flat-bottomed glass vessels containing 500 mL of the
dissolution medium (sink conditions). The vessels were stirred
at 110 rpm and maintained at 32C using an orbital incubator
(Gallencamp Incubator Type INR 2000, Leicestershire, UK). At
appropriate time intervals, 2 mL of samples were withdrawn
and replaced by fresh dissolution medium, then filtered through
a cellulose membrane filter (Filter Paper Whatman 41, GmbH,
Dassel, Germany).
Preparation of Gels
Retinoic acid, in free form and intercalated into HTlc, was formulated as gel formulation according to Farmacopea Ufficiale
Italiana (F.U. XII Ed.) in the section of Semisolid Formulations
Intended for Skin Application. The gel components were mixed
with different order depending of the form of RET: free or intercalated into HTlc. In the former, RET was solubilized in an
ethanol and propylene glycol mixture, diluted with water and
warmed in a steam water bath. The resulting solution was then
added to HEC under continuous stirring (see Table 1). In the
latter, HTlc-RET hybrids were directly mixed to HEC and then
water and ethanol/propylene glycol mixture was added under
continuous and vigorous stirring.
Gel containing free RET (0.05 g) contained ethanol 96%
(20.00 g), propylene glycol (25.00 g), HEC (5.00 g), and deionized water until getting to 100.00 g. Gels containing MgAlHTlc-RET (0.084 g) or ZnAl-HTlc-RET (0.110 g) had the same
composition of gel containing free RET. RET content is the same
of gel containing RET in free form (0.05 g) and the intercalation
products amount was calculated on the basis of drug loadings.
Rheological Studies
Viscometry measurements (viscosity and yield stress measured at 25C and 32C) of formulations were performed by
a Stresstech HR (Reologica Instruments AB, Milano, Italy)
rheometer with cone-plate geometry (diameter of 40 mm, angle
1). Samples were carefully applied to the lower plate using a
spatula to avoid formulation shearing and air bubble formation.
Photostability Studies
Absorption spectra of the solid samples were recorded by
a Varian (Cary 4000) spectrophotometer, equipped with a
150-mm integration sphere and a barium sulfate tablet was
used as reference. The spectra were analyzed by the KubelkaMunk equation in order to make the comparison possible.
The fluorescence spectra and the traces of intensity versus
irradiation time for all the samples were recorded through a
fluorimeter (Spex Fluorolog2) using a front face configuration
Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

Preparation and Characterization of RET Intercalation products


MgAl-HTlc-RET and ZnAl-HTlc-RET were prepared and characterized as reported in Conterosito et al.16 Here, the main
features of the obtained hybrids will be briefly recalled. The
X-ray diffraction analysis (XRPD) demonstrated that the typical reflection corresponding to the pristine nitrate phase
disappears in both intercalation product patterns, mean(8.9 A)
ing that complete substitution of nitrate ions by RET anions
took place. As a result of the RET intercalation, the HTlc inter for MgAllayer distance increased, from 8.9 to 31.7 and 30.5 A
HTlc-RET and ZnAl-HTlc-RET, respectively (data not shown).
The amount of loaded RET in each intercalation product was
determined by thermogravimetric analysis (TGA) after samples equilibration at 75% RH. The obtained thermal profiles
(figures can be found in Supporting Information, SI 1 and SI
2) revealed that the samples underwent a continuous weight
loss as the temperature increased. The first loses until 180C
were attributed to hydration water; in the temperature range
220C500C, the exothermic weight loss (DTA curve) was assigned to the organic moieties oxidation.
Successively, the ICP analysis allowed to measure the content of each metal, Mg, Zn, and Al in both HTlcs. Combining
data coming from ICP analysis and TGA, the molecular formulas of both precursor and intercalated HTlcs were determined.
In the case of MgAl-HTlc in nitrate form, the formula was
[Mg0.66 Al0.34 (OH)2 ](NO3 )0.34 0.4H2 O; its corresponding intercalation product formula was [Mg0.66 Al0.34 (OH)2 ] (RET)0.34 0.66
H2 O, presenting a drug loading percentage (w/w) of 58.9%.
In the case of ZnAl-HTlc in nitrate form, the formula was
[Zn0.70 Al0.30 (OH)2 ] (NO3 )0.30 0.4H2 O; its corresponding intercalation product formula was [Zn0.70 Al0.30 (OH)2 ] (RET)0.30 1.1
H2 O, presenting a drug loading percentage (w/w) of 45.5%.
Differential scanning calorimetry thermal profiles of the hybrids were carried out (figures can be found in Supporting Information, SI 3 and SI 4). Both HTlcs resulted stable in the
temperature range in which the analysis was performed (25C
200C), whereas at higher temperatures (350C480C), the
materials have an endothermic transition because of the loss
of interlayer anions and to the layers dehydroxylation.19 The
thermal profile of crystalline RET (line c) shows a fist endothermic peak at 150C, because of a solid-state transformation of
RET, that is, the monoclinic-triclinic transition20 and a second
at 186C associated to crystals melting.21 This peak can also be
observed in the physical mixtures of HTlc and RET (line b) but
not in the thermal profile of hybrids (line a). This suggests that
the crystalline structure of RET is maintained in the RET/HTlc
physical mixture and loses when RET is intercalated between
HTlc lamellae in which is homogeneously dispersed. On the
DOI 10.1002/jps.24612

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

basis of the chemical composition and the interlayer distance


of both intercalation products, a computer simulated model was
obtained by the HyperChem program (HyperchemTM , 2000) for
MgAl-HTlc-RET and ZnAl-HTlc-RET (figure can be found in
Supporting Information, SI 5). In both cases, the anions organize themselves in order to establish BB interactions among
the double bonds of the polyenic chain. The interlayer distances
are compatible with the ions disposition in the interlayer region
to form a bilayer of molecular anions. In particular, the chains
of the molecular anions are tilted respect to the HTlc planes
forming angles of 40.
In order to evaluate the morphology of both HTlc precursors and the corresponding intercalation product, SEM analyses were carried out on the samples. In regard to MgAl-HTlcNO3 , the micrographs (Fig. 1) show that the crystals have a
regular, hexagonal, and flat shape. Moreover, the crystals show
regular and homogeneous dimensions with a diameter below
2 :m. The corresponding intercalation product MgAl-HTlcRET maintains the same size and morphology of the precursor
as shown in Figure 1, meaning that the intercalation process
does not modify the structure of the lamellar matrix.
The micrographs of ZnAl-HTlc-NO3 (Fig. 2) show crystals
with irregular shape and dimensions. In particular, two different dimensional populations can be distinguished, the first
represented by big crystals with a diameter in the range of 5
8 :m and the second one given by smaller crystals (1.53 :m)
with an irregular shape that probably comes from the breakage
of the bigger ones. ZnAl-HTlc-RET (Fig. 2) maintains the same
characteristics of the precursor and two populations can also
be distinguished in this case.
RET Release Capability from HTlc-RET Compounds
This assay was performed with the aim to evaluate the capability of both hybrids to release the intercalated RET. As
reported in the Materials and Methods section, a mixture of
K2 CO3 0.025 M with the organic solvent THF in the ratio 70:30
was used as dissolution medium. The use of the latter was
necessary as cosolvent because of the low RET water solubility (0.21 :M).22 The choice of K2 CO3 0.025 M solution,18 as
aqueous component of the mixture, comes from the necessity to
simulate above all the air exposure, when the product is placed
on the skin surface.
Both intercalation products, ZnAl-HTlc-RET (220 mg) and
MgAl-HTlc-RET (170 mg), containing an amount of RET (drug)
corresponding to 100 mg of the free form, were placed in two different closed flat-bottomed glass vessels containing 500 mL of
the dissolution medium (sink conditions) and stirred at 110 rpm
at 32C. Initially, the detection of RET released from the hybrids
was performed by UV spectrophotometry; however, this method
did not result suitable because of some limitation associated
both to the chemical nature and low solubility of the molecule.
As clearly reported in literature, when RET is in a water solution, it tends to form aggregates (such as tail-to-tail dimers) or
micelles that present a different spectrum in comparison to that
deriving from free molecules in solution.23,24 Thus, an alternative method was adopted to confirm the effective RET deintercalation from hybrids. Namely, it was decided to recover the
hybrid from the dissolution medium by centrifugation and to
collect its X-ray pattern. By this analysis, RET release was monitored by observing the possible changes of HTlc interlayer distance. As RET release takes place by ion exchange mechanism
DOI 10.1002/jps.24612

3907

with carbonate ions, the interlayer distance should decrease


of the HTlc in carbonate form. Recovering the
from 31.8 to 7.6 A
samples, ZnAl-HTlc-RET and MgAl-HTlc-RET, after 15 min,
the XRPD of both solids showed the peak relative to carbonate
and no signal attributable to RET presence in the HTlc galleries
(X-ray spectra not reported). This means that RET release from
hybrids takes place easily after the contact with the application
site.
Gel Formulation Manufacturing
The choice of the best formulation for HTlc-RET topical administration was performed taking into account the characteristics
that a product intended for skin application must exhibit as
fluidity, spreadability, easy extrusion, capability to be applied
on skin surface by means of a soft massage obtaining a homogeneous distribution of the active molecule. In the case of the
hybrids MgAl-HTlc-RET and ZnAl-HTlc-RET, the presence of
lamellar matrices should be useful because the activity of HTlc
as rheological agents is well documented.25,26 Thus, taking into
account also this aspect, numerous attempts were made in order to prepare a suitable gel for hybrids formulations.
In order to avoid RET deintercalation from the hybrids,
it was necessary to design a gel composition free from ions,
namely, without ions dissolved in the formulation, and using
degassed water. This means that pH-sensitive polymers could
R
) as their gelification takes place
not be used (e.g., Carbopol
in alkaline solutions. Taking into account all these aspects
and considering the composition of hydrophilic formulations for
acne treatment available on the market, simple gels were prepared using cellulose derivatives as gelling agents (polymers
that swell independently from pH value).
Different compositions have been tested using two kinds
of polymers, HEC and carboxymethyl cellulose, many wetting
agents and their different ratios. After various attempts, the
following composition has been selected: ethanol 96% (20.00 g),
propylene glycol (25.00 g), HEC (5.00 g) deionized water until
getting to 100.00 g (Table 1).
The gel was prepared by gentle dispersion of HEC in water, and then ethanol and propylene glycol were added. The
gel containing free RET (0.05%) has been previously solubilized in the ethanolpropylene glycol mixture heated at
low temperatures (about 40C). In the case of HTlc-RETbased gels, the intercalation products were mixed to the polymer (HEC) before water addition (degassed water), and then
ethanolpropylene glycol mixture was added. The compositions of the prepared gels are reported in Materials and Methods section. MgAl-HTlc-RET (0.084 g) (corresponding to 0.05
g of RET because of drug loading 58.9%) and ZnAl-HTlc-RET
(0.110 g) (corresponding to 0.05 g of RET because of drug loading 45.5%) were used (Table 1). In both cases, RET content
(w/w) corresponds to 0.05% as in the case of the commercially
R
gel (contains hydroxypropilavailable formulation Isotrex
cellulose 3% as gelling agent and ethanol) used as product
reference.
After preparation, XRPD analysis has been carried out on
the gels containing the intercalated RET that confirmed the
presence of lamellar structures in the formulations.
Rheological Measurements
Viscometry studies (viscosity and yield stress) were carried
out in order to investigate the flow properties of the prepared
Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

3908

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 1. Scanning electron micrographs of MgAl-HTlc nitrate (a, b) and MgAl-HTlc-RET (c, d).

gels. Measurements were performed at 25C (Fig. 3) and 32C


(Fig. 4) in order to simulate storage and application conditions,
respectively. With the aim to understand the influence of intercalated and not intercalated RET on gel flow properties, the
studies have been performed by using gels containing MgAlHTlc-RET (named MgAl-RET gel), ZnAl- HTlc-RET (named
ZnAl-RET gel), not intercalated RET (named RET gel), an
empty gel (named RET free gel), and the market formulation
R
gel.
Isotrex
All rheograms show that the prepared gels and the comR
present pseudo-plastic behavior
mercial formulation Isotrex
in both temperature conditions (Figs. 3 and 4). Comparing the
rheograms at 32C and 25C, it is clear that, increasing the
temperature, viscosity decreases for all formulations except for
R
(Figs. 3 and 4) that is the most viscous formulation.
Isotrex
On the contrary, it is possible to note that empty gel shows the
lowest viscosity at 25C and 32C. This can be explained considering the absence of the active ingredient (RET) and then the
lack of interactions between RET and HEC polymeric network.
These interactions in fact, responsible for gel viscosity increase,
in this case are absent. As clearly reported in the corresponding rheogram, RET gel shows higher viscosity in comparison to
empty gel to both temperatures.
The most important aspect that must be underlined is the
MgAl-RET gel and ZnAl-RET gel behavior. Figure 3 shows that,
at 25C, the gels containing intercalated RET possess an intermediate viscosity (MgAl-RET gel >ZnAl-RET gel) between
R
gel and the other two formulations (RET gel, empty
Isotrex
gel). This means that HTlc lamellar structure is able to establish numerous bonds with the polymeric network, increasing
the viscosity as well as the formulation texture. This aspect
is very important as by using HTlc in a semisolid formulation
it is possible to increase the gel structure stability during the
storage conditions as well as its shelf-life.
Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

MgAl-RET gel viscosity is higher than ZnAl-RET gel and


this is explainable on the basis of reduced particle size of MgAlHTlc-RET in comparison to ZnAl-HTlc-RET, although high aluminum charge density cannot be excluded.
These properties increase the possibility to form bonds between HEC network and HTlc crystals giving rise to a reinforced gel network structure. Comparing Figure 4 (32C) with
Figure 3 (25C), an interesting phenomenon can be observed.
As a consequence of a limited temperature increase (+7C),
MgAl-RET gel and ZnAl-RET gel viscosity are subject to an
appreciable decrease resulting in lower than RET gel and similar to that of empty gel. This pseudo-plastic-tixotropic behavior
is more evident in the case of MgAl-RET gel, and it can be
explained considering numerous studies reported in literature
in which lamellar hydroxide mixtures of magnesium and aluminum are used as rheological agents because of their ability
to modify the material flow properties.27 These properties are
very important as they are responsible for a better formulation
application and spreadability on skin surface.
Further studies have been made with the aim to evaluate extrudibility (yield stress) of each formulation. This assay has the
objective to evaluate the formulation attitude to squeezability
(ability to take an amount from the package) and spreadability.
These studies have been performed at 25C in order to simulate
the room temperature conditions (storage); yield stress value
comes from the evaluation of viscosity change versus shear
R
shows a different
stress. The commercial formulation Isotrex
behavior, compared with prepared gels, requiring high shear
stress values (1104 Pa) to start its flow (graph not reported).
This is explained considering the high viscosity values registered in previous experiments. Rheograms of prepared gels
(Fig. 5) show that those containing RET (in free form or intercalated into HTlcs) flow at low shear stress values. Particularly
MgAl-RET gel starts to flow at lowest shear stress values than
DOI 10.1002/jps.24612

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

3909

Figure 2. Scanning electron micrographs of ZnAl-HTlc nitrate (a, b) and ZnAl-HTlc-RET (c, d).

Figure 3. Rheograms registered at 25C.

RETH gel and ZnAl-RET gel because of Mg presence that is


able to enhance the flow properties of this formulation. Empty
gel requires high shear stress values to start to flow (about
5103 Pa), showing highest rigidity. Results obtained from rheological studies reveal the advantages in the introduction of
lamellar materials in semisolid formulations. In fact, the studies reveal that the presence of lamellar structure is able to
improve the flow characteristics of the formulation and MgAlHTlc presents the best performances.

DOI 10.1002/jps.24612

Stability Studies
Retinoic acids, such as tretinoin and isotretinoin, are photolabile compounds, whose photostability strongly depends on
the environment.28 This instability occurs even in commercially available product. Absorption spectra of an isotretinoin
formulation before and after steady-state irradiation were in
fact carried out (figure can be found in Supporting Information, SI 6). Absorption intensity decrease (almost 70%) was

Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

3910

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 4. Rheograms registered at 32C.

registered upon 1 h of irradiation testifying the occurrence of


photodegradation processes. To overcome this inconvenience,
intercalation of isotretinoin can be carried out.
Figure 6 reports the UVVis absorption spectra of RET and
its intercalation compounds. The absorption spectrum of RET
has a maxima at 220 nm and shows a broad band in the 350
460-nm range. The intercalation compounds (ZnAl-HTlc-RET
and MgAl-HTlc-RET) present a narrower spectrum with an intense band centered at 330 nm. The broader shape observed for
RET is likely because of BB interactions between chromophore
molecules that might be favored by the packing in the crystalline form. The narrowing of the absorption spectra observed
for the compounds of intercalation confirms the dispersion of
RET in the lamellae.

To evaluate the effects of the inorganic matrices on the photochemical stability of RET and its gel formulations, fluorescence methods were used. Generally, fluorescence is a very sensitive and selective parameter, whose intensity is proportional
to the concentration of the emitting species, thus quite useful to compare the relative photostability of samples measured
in the same experimental conditions. In Figure 7, the fluorescence spectra of RET, MgAl-RET, and ZnAl-RET are reported;
all the samples presented a broad spectrum with maximum
around 550 nm. The effect of steady-state irradiation on the
concentration of isotretinoin samples was quantitatively monitored acquiring the fluorescence intensity at the maximum
upon irradiation and 366 nm. The traces, reported in Figure 7,
indicate that intercalation compounds are more stable than the

Figure 5. Yield stress of the prepared gels registered at 25C.


Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

DOI 10.1002/jps.24612

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Figure 6. Absorption spectra of crystalline RET (squares), MgAlHTlc-RET (circles) and ZnAl-HTlc-RET (triangles).

free RET sample. These data clearly reveal that upon intercalation, both in MgAl-HTlc and ZnAl-HTlc, the drug under investigation improves its photostability, likely because the anchoring
to the matrices reduces the geometrical rearrangements, which
are responsible for the degradation of RETs.
The gel formulations, obtained by homogeneous dispersion
of RET, MgAl-HTlc-RET, and ZnAl- HTlc-RET in gel, have
been tested with the same methods and conditions to evaluate whether their dispersion can modify the sample solidity.
RET and MgAl-RET gels maintain the reached photostability,
as shown by the fluorescence traces reported in Figure 8. On
the contrary, ZnAl-RET gel presents a lower solidity to light
exposure, which needs to be further investigated.

Figure 7. Fluorescence intensity spectra as function of irradiation


time (8irr = 366 nm) of powder samples recorded from crystalline
RET (squares open symbol), MgAl-HTlc-RET (circles open symbol), and
ZnAl-HTlc-RET (triangles open symbol).
DOI 10.1002/jps.24612

3911

Figure 8. Normalized fluorescence spectra as function of irradiation


time (8irr = 366 nm) of gel preparations (open symbols) recorded from
formulations containing crystalline RET (squares full symbol), MgAlHTlc-RET (circles full symbol), and ZnAl-HTlc-RET (triangles full
symbol).

CONCLUSIONS
Retinoic acid has been intercalated into HTlc lamellar structure with the aim to increase its photostability. It showed
high affinity for both MgAl-HTlc and ZnAl-HTlc and its intercalation has successfully occurred giving rise to hybrids,
MgAl-HTlc-RET and ZnAl-HTlc-RET, having high drug loading (58.9% and 45.5%, respectively). In this supramolecular arrangement, the active ingredient loses its crystalline structure
and its molecules are homogeneously dispersed and immobilized into the HTlc nanogalleries.
Intercalated compound crystals are flat and have micrometric size; they can be directly used for the preparation of a
formulation, safe and no-abrasive, suitable to carry the drug
on acneic skin.
Photochemical studies highlighted interesting aspects and
the most important information was obtained from fluorescence
measurements. The fluorescence traces were recorded to have
information about the effect of intercalation into HTlc on RET
photostability. The results clearly reveal that upon intercalation in MgAl-HTlc or ZnAl-HTlc, the active ingredient shows
improved photostability. The formulation obtained by dispersing MgAl-HTlc-RET in gel preserved the obtained degree of
photostability.
Rheology studies showed that the formulations containing
the hybrids are characterized by suitable viscosity and extrudibility, better than commercial formulation and better for MgAlRET. HTlc presence in the formulation furnishes a positive contribution because it confers pseudo-plastic-tixotropic behavior
to the gel, more evident in the case of MgAl-RET. This phenomenon is undoubtedly important for the success and acceptability of final formulation because the increased viscosity at
25C assures product homogeneity with good stability during
the shelf-life (e.g., reduction of sedimentation rate or no sedimentation), whereas the increase of fluidity at 32C can certainly guarantee a good spreadability on the acneic skin injured
and often inflamed.
Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

3912

RESEARCH ARTICLE Pharmaceutics, Drug Delivery and Pharmaceutical Technology

On the basis of all the obtained results, it is possible to state


that RET intercalation into HTlc represents a successful strategy for the preparation of topical formulations safe and able
to avoid all the problems related to RET photochemical instability, mainly in the case of MgAl-HTlc-RET. The inorganic
matrix HTlc, MgAl-HTlc more than ZnAl-HTlc, is able to enhance different properties of the final product. In addition to
carrier and photoprotective activities, in fact, HTlc also improves gel flow characteristics and allows to obtain a very good
dermatological formulation both for safety and for patients
compliance.

ACKNOWLEDGMENTS
The Authors wish to thank Dr. Michele Sisani for collaboration
in the measurements of XRPD and Mr. Marco Marani for the
precious collaboration and technical assistance.
A part of this work was presented at XLIX Symposium AFI,
Rimini (Italy).29
The authors declare that they have no personal financial or
nonfinancial conflicts of interest in this research.

REFERENCES
1. Braun-Falco O, Plewig G, Wolff HH, Burgdorf WHC. 2000. Diseases
of the sebaceous glands. In Dermatology; Heidelberg WB, Ed. 2nd ed.
Berlin Heidelberg, Germany: Springer.
2. Oberemok SS, Shalita AR. 2002. Acne vulgaris, I: Pathogenesis and
diagnosis. Cutis 70:101105.
3. Burkhart CG, Burkhart CN, Lehmann PF. 1999. Acne: A review of
immunologic and microbiologic factors. Postgrad Med J 75:328331.
4. Farrar MD, Ingham E. 2004. Acne: Inflammation. Clin Dermatol
22:380384.
5. Sansone G, Reisner RM. 1971. Differential rates of conversion of
testosterone to dihydrotestosterone in acne and in normal human skina possible pathogenic factor in acne. J Invest Dermatol 56:366372.
6. Makrantonaki E, Zouboulis CC. 2007. Testosterone metabolism to
5alpha-dihydrotestosterone and synthesis of sebaceous lipids is regulated by the peroxisome proliferator-activated receptor ligand linoleic
acid in human sebocytes. Br J Dermatol 156:428432.
7. Haider A, Shaw JC. 2004. Treatment of acne vulgaris. Clin Rev
292:726735.
8. Rigopoulos D, Ioannides D, Kalogeromitros D, Katsambas AD. 2004.
Comparison of topical retinoids in the treatment of acne. Clin Dermatol
22:408411.
9. Leyden J, Grove G, Zerweck C. 2004. Facial tolerability of topical
retinoid therapy. J Drugs Dermatol 3:641651.
10. Perioli L, Ambrogi V, Bertini B, Ricci M, Nocchetti M, Latterini L,
Rossi C. 2006. Anionic clays for sunscreen agent safe use: Photoprotection, photostability and prevention of their skin penetration. Eur J
Pharm Biopharm 62:185193.

Perioli et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:39043912, 2015

11. Perioli L, Ambrogi V, Rossi C, Latterini L, Nocchetti M, Costantino


U. 2006. Use of anionic clays for photoprotection and sunscreen photostability: Hydrotalcites and phenylbenzimidazole sulfonic acid. J Phys
Chem Sol 67:10791083.
12. Perioli L, Nocchetti M, Ambrogi V, Latterini L, Rossi C, Costantino
U. 2008. Sunscreen immobilization on ZnAl-hydrotalcite for new cosmetic formulations. Micropor Mesopor Mater 107:180189.
13. Bish DL. 1980. Anion exchange in takovite: Applications to other
hydroxide minerals. Bull Mineral 103:170175.
14. Miyata S. 1983. Anion-exchange properties of hydrotalcite-like compounds. Clays Clay Miner 31:305311.
15. Costantino U, Marmottini F, Nocchetti M, Vivani R. 1998. New
synthetic routes to hydrotalcite-like compoundsCharacterisation
and properties of the obtained materials. Eur J Inorg Chem 10:
14391446.
16. Conterosito E, Croce G, Palin L, Pagano C, Perioli L, Viterbo D,
Boccaleri E, Paul G, Milanesio M. 2013. Structural characterization
and thermal and chemical stability of bioactive molecule-hydrotalcite
(LDH) nanocomposites. Phys Chem Chem Phys 15:1341813433.
17. Hypercube, Inc. 2000. HyperChemTM , release 6.01 for Windows,
molecular modeling system, Ontario, Canada.
18. Nakayama H, Wada N, Tsuhako M. 2004. Intercalation of amino
acids and peptides into MgAl layered double hydroxide by reconstruction method. Int J Pharm 269:469478.
19. Kannan S, Velu S, Ramkumar V, Swamy CS. 1995. Synthesis and
physicochemical properties of cobalt aluminium hydrotalcites. J Mat
Sci 30:14621468.
20. Berbenni V, Marini, Bruni G, Cardini A. 2001. Thermoanalytical
and spectroscopic characterisation of solid-state retinoic acid. Int J
Pharm 221:123141.
21. Caviglioli G, Pani M, Gatti P, Parodi B, Cafaggi S, Bignardi G. 2006.
Study of retinoic acid polymorphism. J Pharm Sci 95:22072221.
22. Szuts EZ, Harosi FI. 1991. Solubility of retinoids in water. Arch
Biochem Biophys 287:297304.
23. Han H, Zimmerman CL, Wiedmann TS. 1998. Spectral properties
and ionization behavior of retinoids. II. Bile salt solutions. Int J Pharm
172:229240.
24. Washington I, Turro NJ, Nakanishi K. 2006. Superoxidation of
retinoic acid. Photochem Photobiol 82:13941397.
25. Hoy EF. 2005. Rheology modified compositions and modification
agents. Patent US 6914081 B2.
26. Choy JH, Choi S-J, Oh J-M, Park T. 2007. Clay minerals and layered double hydroxides for novel biological applications. Appl Clay Sci
36:122132.
27. Fraser LJ, Burba JL. 1991. Pseudoplastic mixed metal layered hydroxide fluid with fluid loss additive and method of use in penetrating
the earth. Patent EP 91310865.0.
28. Ioele G, Cione E, Risoli A, Genchi G, Ragno G. 2005. Accelerated
photostability study of tretinoin and isotretinoin in liposome formulations. Int J Pharm 293:251260.
29. Perioli L, Ambrogi V, Pagano C, Massetti E, Nocchetti M, Rossi C.
2009. Studi preformulativi di geli a base di acido retinoico immobilizzato in strutture lamellari. 49th AFI Symposium -Rimini (Italy) June
1012, 2009 (abstract page 196).

DOI 10.1002/jps.24612