FACULTY OF PHARMACY DEPARTMENT OF PHARMACEUTICAL TECHNOLOGY AND SOCIAL PHARMACY
DONATAS GRINA
Master thesis was accomplished at a Department of Pharmaceutical Technology, Medical University of Gdansk
Heads of thesis: Prof. hab. dr. Magorzata Sznitowska
Department of Pharmaceutical Technology, Medical University of Gdansk Prof. dr. Vitalis Briedis
Department of Pharmaceutical Technology and Social Pharmacy, Lithuanian University of Health Sciences
KAUNAS, 2011
1.2. The aim of the study............................................................................................................................ 9
2.2. Characterization of consistency......................................................................................................... 12
2.3. In Vitro release test for semisolid dosage forms ............................................................................... 15
2.3.1. Diffusion cells ............................................................................................................................ 16
2.4. Tolnaftate properties.......................................................................................................................... 19
3.1.1. Equipment .................................................................................................................................. 20
3.1.2. Materials ..................................................................................................................................... 21
3.1.3. Reagents ..................................................................................................................................... 22
3.2. Solubility studies ............................................................................................................................... 23
3.2.1. Solubility in organic solvents and solutions of surface active substances ................................. 23
3.2.2. Solubility in oils ......................................................................................................................... 24
3.3. Preparation of semisolids .................................................................................................................. 24
3.3.1. Oleogels...................................................................................................................................... 24
3.3.2. Organogels.................................................................................................................................. 25
3.5. Microscope observation .................................................................................................................... 32
3.7. pH determination............................................................................................................................... 32
3.11. Stability studies ............................................................................................................................... 36
Keywords: tolnaftate, semisolids, developement, analysis, characterization, stability
Tolnaftate (O-2-Naphthyl m, N-dimethylthiocarbanilate) is a topically used antifungal agent,
most commonly employed in the treatment of Tinea Pedis at 1% (w/w) concentration. In the global market
tolnaftate is available in form of solutions, aerosols, powders and creams (in form of suspension), while no
semisolid dosage forms with dissolved tolnaftate are available. Thus in the present study efforts were
made to formulate stable semisolid preparations containing 1% of tolnaftate. It was attempted to formulate
preparations where tolnaftate is dissolved. Tolnaftate is highly lipophilic (LogP 5.6), therefore it was
necessary to evaluate its solubility. Solubility was evaluated in organic solvents, aqueous solutions of
surface active substances, polyethylene glycols and oils. On the basis of solubility studies oleogels,
hydrophilic non-aqueous gels and macrogol ointments were prepared. These preparations were compared
with creams (O/W), where tolnaftate was suspended in the vehicle. Formulation characteristics, such as
spreadability, rheological and mechanical properties, are the most important factors in the development of
semisolid formulations. These tests were carried out with a purpose to select formulations for stability
evaluation. It was impossible to achieve 1% (w/w) tolnaftate concentration in prepared oleogels and
organogels. A solution was successfully obtained in macrogol ointments, however ointment chosen for
stability evaluation was found to be instable during 2 months storage, probably due to interaction between
tolnaftate and polyethylene glycols. Cream (o/w) formulated with triglycerides (Precirol®) and 70% of
water was found to have suitable organoleptic properties, stable semisolid consistency and did not undergo
any significant changes during 2 month storage in 4oC. The cream should be further investigated in long
term stability studies.
Tolnaftatas (O-2-Naphthyl m, N-dimethylthiocarbanilate) yra 1% (m/m) koncentracijoje išoriškai
vartojamas priešgrybelinis preparatas, efektyviai gydantis pd grybel. Tolnaftatas gaminamas tirpal,
aerozoli, milteli, suspensini krem vaist formose, tuo tarpu nra joki pusiau kiet preparat, kur
tolnaftatas bt ištirpintas. Todl siekiama tok preparat sukurti. Šio darbo tikslas – sukurti stabilias
pusiau kietas vaist formas tolnaftatui. Šiame darbe buvo siekiama sukurti puskietes sistemas su ištirpintu
tolnaftatu. Kadangi tolnaftatas stipriai hidrofobin mediaga (LogP 5,6), buvo nustatytas jo tirpumas
organiniuose tirpikliuose, paviršiui aktyvi mediag vandeniniuose tirpaluose, polietileno glikoliuose bei
augaliniuose aliejuose. Remiantis tirpumo rezultatais buvo modeliuojami oleogeliai, hidrofiliniai
bevandeniai geliai ir makrogoli tepalai. Šios vaist formos buvo palygintos su kremais (a/v), kuriuos
tolnaftatas vestas suspensijos pavidalu. Sklaida, reologins bei mechanins charakteristikos yra vieni
svarbiausi veiksni takojani pusiau kiet vaist form kokyb. Remiantis šiomis savybmis buvo
atrinktos puskiets sistemos stabilumo tyrimui. Sukurti 1% tolnaftato tirpalus oleogeli ir hidrofilini
bevandeni geli sistemose nepavyko. Tolnaftatas ištirpo makrogoli tepaluose 1% koncentracijoje.
Stabilumo tyrimui parinktas tepalas nebuvo stabilus dviej menesi laikotarpyje visose tirtose slygose
(4oC, 25oC, 40oC), galimai dl tolnaftato ir polietileno glikoli sveikos. Kremas (a/v) su trigliceridais
(Precirol®) ir 70% vandens turjo puikias organoleptines savybes ir stabili pusiau kiet konsistencij.
Jame dviej mnesi laikotarpyje laikant 4oC temperatroje joki reikšming pokyi nebuvo nustatyta.
Siekiant garantuoti stabilum, kremo tyrimas turi bti tsiamas ilgesn laik.
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ACKNOWLEDGMENTS
It is a pleasure to thank those who made this thesis possible. I owe my deepest gratitude to
dr. Katarzyna Centkowska for her excellent advice, guidance, reading of manuscript, patience and support
which enabled me to develop an understanding of the subject and accomplish this work. I could not have
imagined having a better advisor and mentor for my research.
I am also very grateful to prof. hab. dr. Magorzata Sznitowska for all the advice, critical reading
of manuscript and the opportunity to accomplish thesis in the Department of Pharmaceutical Technology,
Medical University of Gdansk, where I received high quality working conditions and material recourses to
carry out this work.
I am deeply thankful to Toma Keutyt who gave me important guidance during my first steps, for
her motivation, advice and important support througout this work. I would also like to thank
doc. dr. Kristina Ramanauskien and prof. dr. Vitalis Briedis whose guidance helped me completing my
work.
Lastly, I would like to thank my family for all their love and encouragement. For my mother who
raised me to who I am now and supported me in all my pursuits. Thanks be to God for the courage to face
the complexities of life and to complete this thesis successfully.
Donatas Grina
PEG – polyethylene glycol;
1.1. Relevance of study
A wide choice of vehicles, ranging from solids to liquids, are available for topical treatment of
fungal diseases. The majority of them comprises of semisolids: ointments, creams, gels etc. Due to their
peculiar characteristics, semisolids can adhere to the application site for sufficiently long periods before
they are worn off. This property helps prolong drug delivery at the application site and allows achieving
the desired therapeutic effect. Semisolid dosage forms are advantageous in terms of ease of application,
rapid formulation, and ability to topically deliver a wide variety of drug molecules.
Tolnaftate is a topically used antifungal agent, having specific and significant fungicidal effect on
Tricophytone, Microsporum and Epidermophytone. It has a history of 50 years of usage and still remains
one of the most potent drugs for treatment of Tinea Pedis, which affects approximately 10% of the
population. The cure rate of Tinea Pedis is around 80%. In the global OTC product market tolnaftate can
be found in several dosage forms: creams, aerosols, powders or solutions. Liquid preparations are
available, on base of polyethylene glycols, ethanol or isopropanol, however there are no semisolid
preparations with dissolved tolnaftate.
Obects of study: tolnaftate and semisolid dosage forms with tolnaftate.
Aim of study: to formulate stable semisolid preparations having 1% (w/w) of tolnaftate and to
evaluate their properties.
1. Evaluation of tolnaftate solubility;
2. Developement of semisolid dosage forms with tolnaftate:
Oleogels;
3. Analysis of properties (spreadability, rheology, texture profile) of prepared dosage forms
and selection of formulations for stability tests;
4. Stability evaluation of selected formulations stored for 2 months under accelerated testing
conditions (40oC/75%RH), controlled room temperature (22.7±4oC/21.6±1.5%RH) and in
refrigerator (4oC/22%RH):
Generally, the majority of topical formulations comprise of semisolid dosage forms, each having
unique characteristics. The first and a very important step in the formulation of semisolid dosage forms is
the selection of an appropriate, suitable vehicle, agreeably with drug’s physicochemical properties and
required therapeutic application. The active ingredient can be incorporated by either dissolving or
suspending it in the base or one of its phases. When a true solution is formed the order in which solutes are
added is unimportant, while for suspensions the order should be considered, since the dispersed substance
can distribute differently depending on to which phase it is added. If the substance is dissolved in the
formulation at a concentration near (or exceeding) to its solubility at any temperature to which the product
might be exposed, it is necessary to determine if the mixture is visually a single homogeneous phase. It is
recommended to examine the solubility by microscopy. [13, 17]
Vehicles also serve as emollients, protective or occlusive dressings, therefore according to the
desired therapeutic properties this should also be taken into consideration. Oleaginous bases provide an
emollient effect and are beneficial for dry, irritated skin. These vehicles form a barrier on the skin and
prevent moisture loss, whereas water-soluble bases do not provide this effect and allow moisture to escape
from the surface. However, water-based vehicles are easier to wash off. These effects have influence on a
patient’s decision. Patients, with mild and short term disease, prefer easy washable products (e.g. o/w
creams), whereas patients with chronic skin diseases prefer formulations having emollient effect. [13, 17,
39]
Semisolid dosage forms are complex formulations with properties depending upon various
factors, such as size of dispersed particles, interfacial tension between the phases, rheology, partition of
the drug between the phases and so forth. All these factors determine the dissolution profile, consistency
and other characteristics [13]. Characterization of semisolid dosage forms usually include measurements
of consistency (spreadability, viscosity, texture profile), pH, dissolution profile and skin permeation.
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2.2.1. Spreadability
The therapeutic efficiency of a formulation depends on its spreading value. The term
spreadability denotes the extent of area to which a semisolid spreads on application to skin or other
surfaces. This feature determines such properties as ease of application on the surface, correct dosage
transfer to the target site, ease of removal from the package, as well as consumer preference. [9, 15].
The main factors effecting spreadability are temperature, consistency of the formulation, the rate
and time of shear produced. In elastomeric materials an existence of a strong negative correlation between
the spreadability and viscosity has been proved in several studies. Furthermore, strong cohesive forces
within a formulation decrease its flowability, thus having a negative effect on spreading. These factors can
be altered during formulation to achieve desired spreading. Rate and time of shear, temperature and site of
application are other factors influencing spreadability. In order to receive reliable data, these factors
should be concerned when performing tests and should be chosen to be comparable when used by
consumer. [15, 24]
Parallel-plate method is the most often used method for spreadability determination. Several
techniques of performing this method are described in literature [15]. Devena et al. [9] measured
spreadability expressed in terms of time taken by two slides to slip off from the sample placed in between
the slides. Similar techniques were used by Sanjay et al. [31] in their study. Spreadability values during
these studies were calculated from this equation:
S=m*l/t, (1)
l - length of slides,
t - time taken to separate the slides.
Contreras et al. [6] measured the diameters of spread after pressing the sample between the plates
and expressed spreadability as area of extent (Fig.1 ). El-Houssieny et al. [11] used similar techniques, but
applied additional weight on the plates and measured the extent after one minute after each addition. Other
methods for measuring spreadability include subjective assessment, master-curve method, in vivo studies
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these are used rarely. [6, 11, 15].
Fig. 1. Spreadability measurement by parallel-plate method
2.2.1. Rheology
Rheology is described as a science of flow and deformation of materials. Rheological
measurements are essential for characterizing ingredients and finished products. In pharmaceutical
sciences, rheological characterization is important for predicting product performance, consumer
acceptance, manufacturing operations, changes upon storage and transportation. For example, toothpaste
should be easily squeezed out of the tube, a cream shouldn’t be too stiff not to complicate its application,
as well mixing of viscous products requires larger amounts of energy during manufacturing. [5, 24, 26,
29].
Deformation is described as a result of force acting on a matter. If this force is tangential it is
described as shear stress, which is expressed in pascals. Shear stress necessary to move, parallel to the
sliding plane, a layer of liquid of 1 square metre at a rate (v) of 1 metre per second relative to a parallel
layer at a distance (x) of 1 metre, is termed as dynamic viscosity or viscosity coefficient. Dynamic
viscosity (Pa*s), is calculated from the following equation: η = τ/D, where D is a speed gradient (dv/dx)
and is termed as rate of shear or shear velocity (s–1). In non-Newtonian systems viscosity is not a
coefficient – it varies with the shear stress and the consistency depends upon the duration and shear rate.
Semisolid formulations require flexibility in drug delivery, therefore a time-dependent change in viscosity
is a desired property. Semisolid dosage forms, being Non-Newtonian systems, can exhibit plastic,
pseudoplastic or dilatant behaviour. [12, 24]
Plastic materials, also termed as Bingham Bodies, require stress to be above the yield value (yield
stress) for flow to be visible in a short term. If yield stress is applied plastic materials exhibit free flowing.
The existence of yield value is explained by Van DerWaals and Born forces, and the formation of three-
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dimensional networks due to concentrated particles. In pseudo plastic materials the flow elements do not
adapt to applied stress instantaneously, as a result shear thinning occurs – viscosity decreases with the
increase of shear rate. When the stress is removed, Brownian motion leads to structure recovery and
increase of viscosity. The gradual decrease in viscosity with applied stress followed by gradual recovery
when the stress is removed is termed as thixotropy. Dilatant materials show increase in viscosity with
increasing the rate of shear. Dilatant flow is typical in pastes and suspensions with high concentrations of
solid particles. [24, 29]
described in various pharmacopeias. European Pharmacopoeia contains a monograph describing the use of
rotating viscometers for measuring rheological properties of non-Newtonian materials. European
Pharmacopoeia classifies rotating viscometers to absolute and relative. Absolute include concentric
cylinder and cone-plate viscometers, relative – spindle viscometers. Other rotating instruments, not
described by Ph. Eu., include parallel plate systems. However literature does not recommend this
geometry for viscosity measurements, because of shear rate variation along the gap. [12, 24].
2.2.3. Texture profile analysis
Texture profile analysis (TPA) is a technique that has been extensively employed to mechanically
characterize food. Recently, it became useful technique in the field of pharmacy for characterizing
semisolid dosage forms. Jones et al. [19, 20] reported TPA as a convenient method in the use of
characterizing consistency of semisolid dosage forms (creams, gels) in several studies. Furthermore,
Coviello et al. [7] verified the potentiality of TPA in the field of pharmacy in their study.
TPA is a penetration/withdrawal experiment when a solid probe is penetrating to a certain depth
and then returns to its starting position. As a result force-time curve is obtained, from which following
parameters are calculated:
Consistency/compressibility – positive area covered by the force-time curve. This value
represents the work needed to overcome the internal bonds of the material;
Cohesiveness/adhesiveness – the maximum force produced on probe withdrawal;
Index of viscosity/work of adhesion – negative area covered by the force-time curve. Value
represents the work needed to pull the probe away from the sample. [7, 35]
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Texture properties have a direct influence on clinical efficacy of the product. Firmness and
consistency are properties that are referred to the ease of removal of the formulation from the container
and ease of application on the substrate. Low values of firmness and consistency would ensure that
minimum work is required to remove the formulation from the container and administer onto the surface.
Cohesiveness and index of viscosity refer to the work required to overcome the attractive bonds between
the sample and the probe, thus determining adhesion on the surface. Bigger values are desired to ensure
prolonged adhesion. [19, 20, 36]
Several factors effecting mechanical characteristics of a product are described in literature. Jones
et al. [19, 20, 21] characterized hydrogels with hydroxyethylcellulose, polyvinylpyrrolidone, and
polycarbophil in several studies and reported the effect of polymer concentration on product texture
characteristics. Tan et al. [36] and Coviello et al. [7] reported same results in their studies – parameters
increased with increasing polymer concentration. Texture characteristics were compared with rheological
properties of agarose gels by Barrangou et al. [1]. This study proved an existence of direct influence of
viscosity on the mechanical parameters defined by textural analysis.
2.3. In Vitro release test for semisolid dosage forms
In vitro release test is one of the several standard methods used to characterize performance of
semisolid dosage forms. It reflects combined effects of several physical and chemical parameters, such as
solubility and particle size of active ingredient, rheological properties etc. Dissolution studies serve as a
research tool in development of formulations. Furhtermore, it can be used to carry out
bioavailability/bioequivalence studies for semisolids to demonstrate quality, efficacy and sameness of the
product upon any changes in excipients, manufacturing process or during storage. However, no validated
method for in vitro release testing could be found in any pharmacopeias. Only recommendations exist in
the guidelines provided from OECD and FDA. [13, 14, 27, 28, 33].
During in vitro release test the kinetics of diffusion of an active ingredient through a semisolid
into acceptor solution are measured. A sample is placed in an open donor chamber and is separated from
the liquid with an inert, porous membrane. Drug diffusion across membrane is monitored by assay of
sequentially collected samples of acceptor solution. Several commercial instruments are available for in
vitro release testing. Elements of release testing to consider involve diffusion cell, membrane, acceptor
medium, temperature and sampling time [13, 40].
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2.3.1. Diffusion cells
Various diffusion cells are described in literature. These include Franz cell, enhancer cell,
insertion cell, Bronaugh flow-through, Plexiglas flow-throug, Hanson ointment cell, and others. Franz and
enhancer cells have been extensively investigated for in vitro release from semisolid dosage forms and
many methods with the usage of these cells could be found in literature. A static one-chamber diffusion
cell described by Franz is recommended by both FDA and OECD. In Franz cell (Fig, 2 (A)) receptor
solution is placed into the receptor compartment which is maintained at 32oC. An artificial support
membrane is placed over the diffusion cell opening. The formulation is applied to the membrane. Cells are
stirred with a magnetic stir bar [4, 13, 17, 27, 33, 40]. Solich et al. [34] in their study successfully
developed a fully automated system for in vitro release testing of semisolid dosage forms based on the
sequential injection analysis technique coupled with the Franz cell. This method was also described by
Klimundova et al. [23]. Both these studies proved that this method is flexible, easily automated, fast and
economic.
An alternative to Franz cell is the enhancer cell (Fig. 2 (B)), introduced by VanKel industries in
early 1990’s. It is used with a modified USP Type II dissolution apparatus, where a 200 ml vessel is used
[4, 25]. This technique was used by Segers et al. [32] for studying the in vitro release of phenol from
ointments. In this method cell rests at the bottom of the vessel with a paddle stirring medium above it.
Study revealed that reproducible and reliable data can be obtained with the usage of enhancer diffusion
cell.
Insertion cell (Fig. 2 (C)) is increasingly used for measuring drug release from semisolid dosage
forms. It was constructed such that its dimensions permitted this cell to be used with the compendial flow-
through cell. Insertion cell is a modified version of USP Type IV dissolution apparatus. Chattaraj and
Kanfer [3] monitored the release of acyclovir from semisolid dosage forms. Obtained values were
favorably compared with the results obtained using Franz diffusion cells. Also insertion cell offers
advantages compared with Franz cell, since it does not require the removal of air bubbles from the
membrane, which is common when using Franz cells. [17, 25]
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Fig. 2. Schematic representations of dissolution apparatuses: A – Franz cell, B – Enhancer cell, C – Flow-through cell [25]
A comparative study between Franz and enhancer cells was performed by Sanghvi and Collins
[30]. It was concluded that enhancer cell is more advantageous when compared to Franz cell: it
demonstrated higher durability, was easier to use and no apparent change was observed in the condition of
the ointment or the skin. Liebenberg et al. [25] compared Franz, enhancer and flow-through diffusion
cells. The main advantages of the Franz and Enhancer cells over the flow-through cells were found to be
the ease of operation and larger sample sizes ensuring more consistent results. Franz and Enhancer cells
gave similar release results for the products tested in this study. It is also stated that enhancer cells are
advantageous when compared to Franz, because they are used in the basic USP dissolution apparatus and
have a larger volume range, making it easier to adapt the system to study the release of products
containing low concentrations of active ingredients or ingredients that are difficult to analyze.
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2.3.2. Membrane and acceptor medium
Membrane and receptor medium should be considered together – they have to be compactable.
Furthermore, since membranes are porous, they absorb the liquid and acceptor medium becomes part of
the membrane. Synthetic membrane provides physical support and prevents bulk transfer. FIP/AAPS
guidelines indicate that sometimes it may be possible to perform the test without a membrane, depending
on the characteristics of the semisolid. The membrane should be highly permeable to the drug and inert for
both semisolid and acceptor medium. Excipients may affect the physical integrity of the membrane or the
active ingredient may bind to the membrane in many cases. Therefore it is recommended to test
membranes for drug binding by passing standard solutions of test compound in the receiving medium of
concentration levels, comparable to those which will be encountered during the experiment. Furthermore,
pre-soaking of the membrane in the acceptor medium is recommended to ensure that air within the pores
is completely replaced with the liquid [33, 37, 40]. However, while developing a release test for retinoic
acid in various semisolid formulations, Thakker and Chern [37] observed that, for retinoic acid
formulations, pre-treatment of the membrane had little or no effect on the dissolution profile.
The most important requirements for acceptor medium is to have good solvent properties and
maintain sink conditions – solubility of the drug should be a minimum of ten times the highest expected
concentration within the liquid. It is also required that acceptor medium would be completely immiscible
with the semisolid vehicle. Another consideration is pH – it should be based on the formulation, pH-
solubility profile of the drug and the pH of the targeted surface [37, 40]. Viegas et al. [38] developed a
dissolution apparatus to evaluate tolnaftate (TOL) release from topical powders, which consisted of a
mesh unit to support the powder, receptor phase and a sink (chloroform). The sink design was employed
to accommodate the inherent low aqueous solubility of TOL. The effect of pH on the dissolution rate was
also examined in this study. Aqueous buffers of pH 3, 5, 7 or 8 were used as the receptor phase. Results
showed that the percentage of drug released increased at low pH levels: the greatest release was observed
in the pH 3 buffer, followed by the pH 5 and pH 8 buffers. However, the dissolution rate at pH 7 was
found to be the lowest.
2.3.3. Temperature and sampling time
Receptor temperature is generally set to 32oC to mimic skin surface temperature. Deviations are
justifiable when the specific action sites are targeted, for example vaginal mucosa, in which case, 37oC is
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more appropriate. It is also recommended that extensively high temperatures, capable of melting the
product or otherwise causing any significant physical changes, should be avoided. [33, 37, 40]
Considering sampling time, a time window during which release experiments should be
performed exists. If the release is quite low, early time values of amount released may not represent the
situation. If the released drug content exceeds approximately 35-45% of the initial content, the
assumptions underlying the equations which support a linear relation between amount released and the
square root of time are no longer valid. Thus sampling times may have to be varied depending on the
formulation and data should be collected between these two extremes: after the influence of membrane
and its associated stagnant layer disappears and before excessive drug depletion from the semisolid
occurs. FIP/AAPS guidelines direct that at least 5 sampling times are needed to generate an adequate
release profile and suggests a 6-hour study period with sampling at 30 minutes, 1, 2, 4 and 6 hours. After
each sampling the removed amount of acceptor medium has to be replaced with a fresh liquid. [33, 40]
2.4. Tolnaftate properties
In 1963 Noguchi and colleagues reported a series of naphthiomates with antifungal activity, of
which TOL (O-2-Naphthyl m, N-dimethylthiocarbanilate) (Fig. 3) was found to be the most promising
compound. TOL acts by selectively inhibiting squalene epoxidase, which results in the accumulation of
squalene and deficiency of ergosterol in the cell walls of fungi. It is available over-the-counter used
topically in concentration of 1% in creams, aerosols, powders or solutions to treat superficial mycoses of
the skin, caused by such fungi as T. rubrum, T. mentagrophytes, T. tonsurans, E. floccosum, T.
schoenleinii, M. canis, M. audouinii, M. gypseum, M. furfur, M. japonica. The cure rate of Tinea pedis
(athlete’s foot) is around 80%. However, TOL is not effective and shouldn’t be used alone against
infections of hair and nails, also it doesn’t affect Candida albicans, Cryptococcus neoformans and most
strains of Aspergillus fumigatus, bacteria, protozoa and viruses. [2, 8, 22]
Fig. 3 Chemical structure of TOL (O-2-Naphthyl m, N-dimethylthiocarbanilate)
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TOL is white to creamy white crystalline odorless powder, which melts at 110-113oC. It has a
LogP equal to 5.6526, and, having strong lipophilic properties, it is freely soluble in acetone and
methylene chloride, very slightly soluble in alcohol, sparingly soluble in ether and practically insoluble in
water (0.0702 mg/l). [8, 18]
Gennaro et al. [16] stated that only occasional burning sensations and maceration were reported
and that these adverse effects were probably caused by endotoxins of killed fungi. TOL has an oral LD50
in mice of >10 g/kg and in rats of >6 g/kg. Furthermore, the substance is not absorbed percutaneously into
the blood stream, because it is too lipophilic to pass hydrophilic viable skin layers, so it can not cause any
systemic effects. [8]
Analytical balance (type WAX 62, Zakad Mechaniki Precyzyjnej, Radwag, Radom, Poland)
Centrifuge (WE-6, Mechanika Precyzyjna, Warsaw, Poland)
Chromatographic column 250 x 4mm, RP C-18, 5 µm (LichroCART/Lichospher 100)
Dissolution apparatus (PharmaTest, type – PTW S3, Hainburg, Germany)
Electronic balance (Sartorius, type PT 600, Germany)
Electronic balance (type WPS 210/C/2, Zakad Mechaniki Precyzyjnej, Radwag, Radom, Poland)
Heating chamber (type KC-65, Premed, Poland)
High performance liquid chromathography (HPLC) system (Merck Hitachi, Darmstadt, Germany)
equipped with UV-Vis detector.
Magnetic stirrer with controlled temperature heater (Type MR 3001 K, Heidolph. Kelhheim,
Germany)
Microscope – type B1 223A (Motic, Welzlar, Germany), equipped with digital camera – type GP-
KR 222 (Panasonic, Osaka, Japan)
Fluorescent microscope (Nikon Eclipse 50i) equipped with digital camera (Nikon Digital Sight
DS-U2) and super high pressure mercury lamp (C-SHG1) (Nikon Corporation, Japan)
pH-meter (Orion, Model 350, Orion Research, Boston, USA)
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Spreadability measuring set (Medical University of Gdansk)
Test tube shaker ”Vortex” (DUN, Warsaw, Poland)
Texture analyzer (TA.XTPlus, Stable Micro Systems, UK)
Unguator e/s (GAKO Konietzko, Bamberg, Germany)
Viscometer (ViscoTester HAAKE, type VT 550, Karlsruhe, Germany)
Water bath equipped with immersion thermostat (EH 4 Basic, IKA, Germany)
Water purification apparatus with ion exchange and reverse osmosis unit (Elix3, Millipore,
Bedford, USA)
3.1.2. Materials
o Orange glass jars (Labart, Poland)
o Orange glass tubes with full cap with PTF laying (American National Can, USA)
o Chromacols (Trumbull, USA)
Goscicino, Poland)
Laboratory materials:
Paraffin film – Parafilm (American National Can, USA)
Semipermeable membrane from cellulose esters (Cuprophan, type 150 pm, MW 10000, Medicell
International, UK)
Texture analyzer probe - back extrusion rig 40 mm disc (type A/BE-d35, Stable micro systems,
UK)
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Castor oil (Pharma Cosmetic, Krakow, Poland)
Cetostearyl alcohol (Sigma-Aldrich-Fluka, USA)
Methylene chloride (Polskie Odczynniki Chemiczne S.A., Gliwice, Poland)
Ethanol 95% (Z.P. Polmos Lublin, Lublin, Poland)
Empiwax - sodium lauryl sulfate and cetostearyl alcohol mixture 1:9 (Przedsibiorstwo
Produkcyjno Usugowe, Poland)
Glycerol (Laboratorium Galenowy, Olsztyn, Poland)
Isopropanol (J. T. Baker, The Netherlands)
Isopropyl myristate 90% (Sigma-Aldrich, USA)
Jojoba oil (Carl Roth, Germany)
Liquid paraffin (Laboratorium Galenowe Olsztyn, Olsztyn, Poland)
Medium chain triglycerides (Miglyol, Nordmann – Rassmann, Warsaw, Poland)
Methanol pure, HPLC grade (POCH SA, Gliwice, Poland)
Miglyol oil (Nordmann – Rassmann, Warsaw, Poland)
Olive oil (Jeronimo Martins Dystrybucja, Kostrzyn, Poland)
Polyethylene glycol 300 (Fluka Chemie, Switzerland)
Polyethylene glycol 300 (Sigma Chemical Co, USA)
Polyethylene glycol 400 (Lancaster Synthesis, UK)
Polyethylene glycol 400 (Sigma-Aldrich Chemie GmbH, Germany)
Polyethylene glycol 1500 (Loba Chemie, Wien – Fischamend, Austria)
Polyethylene glycol 1500 (Merck, Germany)
Polyethylene glycol 4000 (Sigma-Aldrich Chemie GmbH, Germany)
Precirol ATO 5 - Glyceryl palmito-stearate (Gattefosse, France)
Rape oil (Pharma Cosmetic, Krakow, Poland)
Silicones for personal care, 334EU Silicon fluid (Dow Corning Europe, Belgium)
Sodium dodecyl sulfate (Fluka Chemie, Switzerland)
Solid paraffin (Laboratorium Galenowe Olsztyn, Olsztyn, Poland)
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Span 80 (Sigma-Aldrich GmbH, Spain)
Span 80 (Fluka, Spain)
Sunflower oil (Vitacorn, Poznan, Poland)
Tolnaftate (Sanitas, Kaunas, Lithuania)
White petrolatum (Laboratorium Galenowe Olsztyn, Olsztyn, Poland)
White wax (PPH Galfarm Sp., Krakow, Poland)
3.2. Solubility studies
3.2.1. Solubility in organic solvents and solutions of surface active substances
TOL samples (10 mg) were placed in test tubes. Dispersion was formed by adding 1 ml of a
solvent and mixing on the vortex for 10 min. Solubility was evaluated visually: if solid particles were still
observed another milliliter of the same solvent was added. These procedures were repeated till a solution
was achieved or 0.1% concentrations were reached. Solvents used are listed in table 1. Organic solvents
and their mixtures with water in different concentrations were used:
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Ethanol ET 95%, 90%, 70%
Polyethylene glycol 300 PEG 300 100%, 90%, 80%
Polyethylene glycol 400 PEG 400 100%, 90%, 80%
Polyethylene glycol 600 PEG 600 100%, 90%, 80%
Propylene glycol PG 100%
Isopropyl myristate IPM 90%, 80%
Chloroform CHL 100%
Tween 60 TW 5%
3.2.2. Solubility in oils
TOL solubility was evaluated for rape, olive, jojoba, Miglyol, flaxseed, and castor oils. Petri
plates were used to heat 1 g of oil on a heating panel with controlled temperature (80oC). A sample of 10
mg of TOL was added to the heated oil and mixed with a glass stick for 15 min. Then a specimen of the
solution was transferred on a microscope slide and observed under the light microscope to evaluate
dissolution. This procedure was repeated till a solution was achieved or 10% concentrations were reached.
Samples were also examined under the microscope after 24 h and after 7 days to observe if
recrystalization occurs.
3.3.1. Oleogels
Oleogels were prepared using different types of oils (Table 2) at 89% (w/w). Oils were heated on
a heating panel, with controlled temperature (80oC). TOL was added at 1% (w/w) concentration and
mixed with a magnetic stirrer till it dissolved. The heating was turned off and Aerosil was gradually
incorporated to a hot solution at 10% (w/w). Dispersion was then cooled and mixed till gelification.
-25-
Table 2. Oils used for oleogel preparation. Type of oil Abbreviation
Castor G1
Avocado G2
Olive G3
Miglyol G4
Jojoba G5
3.3.2. Organogels
TOL was dissolved in 60oC hot PEG 400 or PG at 1% (w/w) concentration. Hydroxypropyl
cellulose (HPC) was added and mixed. The solution was cooled and ethanol was added. Organogel bases
were prepared according to compositions shown in table 3.
Table 3. Organogel base composition (% w/w). Component OG 1 OG 2 OG 3 OG 4
HPC 3 5 5 5
Ethanol 95% 30 30 30 20
PEG 400 66 64 - -
PG - - 64 74
3.3.3. Macrogol ointments
Ointments were prepared by mixing different types of macrogols (Table 4). Solid PEG was
melted on a heating panel with controlled temperature (60oC) and mixed with the liquid PEG. TOL was
dissolved in the mixture of polyethylene glycols at 1% (w/w) concentration. The heating was turned off
and the solution was mixed mechanically till it became semisolid. Macrogol bases were prepared
according to compositions shown in table 4.
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Table 4. Macrogol ointment base composition (% w/w). Component MO 1 MO 2 MO 3 MO 4 MO 5
PEG 300 - - 30 80 -
PEG 1000 - 50 70 - -
PEG 1500 50 - - - 40
3.3.4. Creams
Cream bases were prepared by heating aqueous and oily phases in separate flasks. Because
isopropyl myristate does not mix with semisolid hydrocarbons it was heated separately with paraffin as a
third phase. All flasks were placed in a heating chamber to reach a temperature of 70oC. Isopropyl
myristate and liquid paraffin were mixed with the oily phase for 5 min at speed level 5 with Unguator.
Then aqueous phase was added and mixed at speed level 1. Mixing was continued till the cream was
homogenic and semisolid. Cream bases were prepared according to compositions shown in table 5.
TOL was incorporated by suspending it in the base. Required amount of TOL was crushed and
grinded with a pestle in a mortar. A small amount of the base was added to the mortar and TOL was
dispersed. The remaining base was transferred to the mortar in several portions with constant mixing in
order to have a homogenic suspension.
-27-
Cream Component
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15
Water 50 60 47 50 50 47 65 64 70 70 70 70 70 70 70
Tween 60 5.0 2.0 3.0 - - 4.0 5.0 5.0 5.0 6.0 5.0 5.0 - - 5.0
Tween 20 - - - - - - - - - - - - 7.5 4.4 -
Cetostearyl alcohol 15 6.0 8.0 15 13 10 12 6.0 8.0 - 8.0 8.0 6.0 8.0 8.0
Span 40 - - - - - - - - - - - - 2.5 3.6 -
Petrolatum 25 20 - - - - - - - - - - - - -
White wax - - - - - - 6.0 - - - - - - - -
Isopropyl myristate - 6.0 - - 5.0 3.0 10 5.0 7.0 5.0 - - 7.0 7.0 7.0
Liquid paraffin 5.0 6.0 15 10 10 15 - 5.0 - - 7.0 - - - -
White paraffin - - 20 - - 15 - - - - - - - - -
Stearic acid - - 4.0 - - 2.0 - 2.0 - - - - - - -
Cholesterol - - 1.0 - - 2.0 - 1.0 - 1.0 - - - - -
Empiwax - - - 10 10 - - - - 4.0 - - - - -
Silicon oil - - - 5.0 4.0 - - - - - - - - - -
Miglyol oil - - - - - - - - - - - 7.0 - - -
3.4. Consistency evaluation
3.4.1. Spreadability studies
Spreadability studies were carried out using a glass disc and a glass plate with a millimeter grade
scale (Fig 3). An opened syringe was used to put 1 cm3 of a sample in the center of the plate. Pre-weighted
glass disc was put on the plate spreading the sample. Diameters of spread circles were measured after 1
min. A flyweight of 200 g was put on the centre of the glass disk and the diameter of spread was again
measured after 1 min. This procedure was repeated by adding another flyweight of 200 g till a weight of
1000 g was reached. The spreadability is considered as area (cm2) covered by the sample. The bigger is
the area, the more spreadable is the sample. Spreadability studies were made in single measurements.
Results were shown as dependence between covered area and the weight applied.
A B C
Fig. 3. Spreadability measuring methodology: A – spreadability measuring set, B – weight applied on a cream, D – measurement of spread area
3.4.2. Rheology studies
Rheology studies were performed according to Ph. Eur. VII. A rotational viscometer
(ViscoTester VT550 Fig.4) was used and a cone-plate method was applied. A small amount (about 300
mg) of ointment was pressed between a rotating cone and a fixed plate, as shown in figure 4. A motor
turned the cone at a steady angular rate. During the measurements samples were thermostated at 20°C.
The following settings were used:
Gap – 0.05 mm
Gels: 0 - 60 1/s, t = 120.00 s
Creams: 0 - 40 1/s, t = 60.00 s
Ointments: 0 - 30 1/s, t = 40.00 s
A B
C
Fig. 4. A – measuring device (cone-plate), B – thermostated apparatus, C – cone-plate scheme
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The collected data was analysed using RheoWin 3.5 Data Manager. Viscosity, tixotoropy and
area under the flow curve were registered. Viscosity was calculated from the following equation:
η – dynamic viscosity [N/m2 s = Pa · s]
τ – shear stress [N/m2 = Pa]
– shear velocity [s -1]
studies were performed in sextuple measurements.
3.4.3. Texture profile analysis
Texture analyses were performed using a TA.XTPlus texture analyzer (Fig. 5 A). Tests were
performed in a plastic ointment container. The container was completely filled with approximately 25 g of
the tested ointment (Fig. 5 C) and stored at room temperature for 24 h for equilibration. The container was
positioned centrally 60 mm below the probe (A/BE-d35) and it was held to prevent it from lifting during
probe return. Texture analyses were performed in triplicate measurements.
A B C
Fig. 5. A – texture analyzer, B – probe position, C – filled container
-31-
Test mode: Compression
Trigger Force: 0.1 g
The probe was moving down at a speed of 1.5 mm/s till a 0.1 g surface trigger was attained. At
this point the probe was in full contact with the sample surface. Then the probe continued to penetrate to a
depth of 10 mm at a speed of 0.5 mm/s. At this point the probe returned to its starting position. A typical
obtained texture plot is shown in figure 6.
0 10 20 30 40 50 60 70 80
0,200
0,175
0,150
0,125
0,100
0,075
0,050
0,025
0,000
-0,025
-0,050
-0,075
-0,100
-0,125
-0,150
Fig. 6. Parameters of typical ointment texture plot
Peak (maximum force) is considered a measure of firmness – the bigger the force the thicker is
the sample. The area under the upper curve is a measure of consistency – the higher the value the thicker
Firmness
Consistency
Cohesiveness
-32-
consistency of the sample. The negative part of the curve is produced on probe return. The maximum
negative force is considered an indication of cohesiveness. The bigger the force the more cohesive is the
sample. The higher value the negative area has, the more resistant to withdrawal is the sample. This is
considered as index of viscosity of the sample. These texture measurements depend on methodology and
equipment used so they can not be compared directly with other rheology parameters, which were attained
for example using a cone-plate method.
3.5. Microscope observation
Polarization microscope was used during the stability studies. Small samples were placed on the
microscope slides and carefully covered with cover glasses. Size (minimum and maximum) and
morphology of crystals were evaluated in suspensions.
3.6. Organoleptic and macroscopic evaluation
Color, odor and consistency were evaluated. A sample was applied on the skin to determine if
the preparation is greasy, sticky, easy washable.
3.7. pH determination
A sample of 1 g was added to a test tube and 9 ml of deionized water was added. After 10 min on
a vortex the mixture was filtrated. Creams were heated at 50oC for 15 min to melt the lipids. Then they
were mixed on a vortex again for 10 min and filtrated. pH was measured in triplicate experiments using
pH-meter.
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3.8. Drug assay
Content of TOL in macrogol ointment was analyzed by applying 0.250 g of an ointment to a test
tube and adding 10 ml of ethanol. The sample was mixed on a vortex for 10 min, diluted with methanol
(40x) and analyzed by HPLC.
Creams were analyzed by applying 0.250 g of a cream to a tightly closed flask and adding 25 ml
of ethanol. The flask was placed in a water bath (50oC) for 30 min. Then it was shaken for 5 min and the
heating was continued for another 30 min. After that samples were placed in a ultrasound bath for 5 min
and stored in a refrigerator for 1 h to solidify the lipids. The samples were centrifugated to seperate the
lipids from the solution. The solution was then diluted with methanol 1 : 1 and analyzed with HPLC.
Control samples were used in order to indicate the efficiency of extraction method. Content analyses were
performed in sextuple experiments.
3.9. Dissolution studies
About 1 g of the ointment was placed in the enhancer diffusion cell. Cuprophan membrane was
presoaked in deionized water. PEG 400 and ethanol mixture (1:1) was used as a receiving medium in a
volume of 50 ml. Dissolution studies were carried out in teflon chambers (VanKel type) in
pharmacopoeial paddle apparatus (PharmaTest, Fig. 7) termostated at 32 ± 1C (stability studies) or
37 ± 1C (preliminary studies). Stirring paddle was lowered to 1 cm above the sample surface and rotated
at a speed of 50 rpm. The glass vessel was tightly covered with aluminum foil and a plastic cap to avoid
evaporation of organic solvents. Samples of 5 ml were taken after 30, 60, 120, 240 and 360 min and were
replaced with a fresh acceptor solution. Dissolution studies were performed in sextuple experiments.
Samples were kept in refrigerator till analyses.
-34-
An UV-Vis spectrophotomety was used in preliminary experiments for quantitive determination
of TOL in solutions: ethanol, diluted dissolution media (10 times with ethanol) and dissolution samples.
The calibration curve was made in range of 1 to 10 µg/ml. The absorbance spectrum (Fig. 8) was
measured between 200 and 310 nm. Two peaks with maximum at 222 and 258 nm were registered.
Dissolution samples were diluted 10 times with ethanol and placed in a 1 cm glass cell for analyses. PEG
400 and ethanol 95% mixture (5:95) was used as a reference.
Fig. 8. Spectrum of TOL solutions in ethanol (concentration range 1-10 µg/ml)
-35-
Fig. 9. UV-Vis calibration curve of TOL in ethanol
Concentrations [µg/ml] after UV-Vis analyses using this calibration curve (Fig. 9) were
calculated from absorbance (A) using following equation:
C = (A – 0.0082) / 0.0706 R2 = 0.9996
3.10.2. High performance liquid chromatography
Quantitive determination of TOL was performed by reverse phase high performance liquid
chromatography (HPLC). Reverse phase C18 5 µm column was used and TOL was detected at 258 nm.
Methanol 80% (v/v) was used as a mobile phase at a flow rate 1 ml/min. The injected volume was 20 µl.
Standard curve (Fig. 11) was obtained within concentration range 1 to 10 µg/ml. The retention time (tR) of
TOL was approximately 9.6 min as shown in figure 10.
Fig. 10: HPLC chromatogram of 10 µg/ml standard TOL solution in methanol
TOL
-36-
Fig. 11. HPLC calibration curve of TOL in methanol
Concentrations [µg/ml] after HPLC analyses using this calibration (Fig. 11) curve were
calculated from area (A) using following equation:
C = (A + 5,3769) / 79,184 R2 = 0.9993
3.11. Stability studies
Macrogol ointment (MO 5) and a cream (C 9) were chosen for stability tests. Amounts of 300 g
of ointment and cream were prepared (points 3.3. and 3.4 Methodology). Ointment and cream were
divided into portions (about 40 g) and stored for certain time in selected temperatures. Stability tests were
performed according to EMEA guidelines. Samples were stored for 2 months in accelerated conditions
(40oC/75%RH), controlled room temperature (22.7±4oC/21.6±1.5%RH) and in a refrigerator
(4oC/22%RH). Tests performed are summarized in table 6. Macroscopic and organoleptic properties were
evaluated during storage for both cream and ointment. A paired t-test (alpha=0.05) was performed with
MS Excel to determine if the difference between the means of obtained results was statistically significant.
-37-
Table 6. Plan of stability tests for ointment and cream ( - test performed). Temperature [oC]
25 4 25 40 Time [months]
Tests
Microscope ob. pH - -
4. RESULTS AND DISCUSION
In the global OTC product market there are solutions of TOL available on the basis of
polyethylene glycols, ethanol or isopropanol, while there are no semisolid preparations with dissolved
TOL. There are also powders, suspensions in creams and sprays available. “Dr. Scholl's Athlete's Foot”
cream is the only preparation of TOL currently available in Lithuania. In the present study efforts were
made to prepare semisolid dosage forms with 1% of TOL dissolved in the base.
TOL solubility in organic solvents
TOL is a very lipophilic compound (LogP=5.6) and is practically insoluble in water – 0.0702
mg/l [18]. It was important to determine solubility of TOL in oils, organic solvents and their mixtures with
water according to point 2 of methodology. Experimental results for organic solvents are presented in
tables 7 and 8. On the basis of obtained results it was proved that TOL is slightly soluble even in organic
solvents, however there were differences between solvents. Chloroform tends to be the best organic
solvent among examined ones, as the highest solubility (20 mg/ml) was achieved in it. There was no
difference between solubility in ethanol and isopropanol (3.33 mg/ml). Even pure ethanol (95%) and
isopropanol can not be used as single solvents in semisolid preparations and topical solutions, since
solubility is in these solvents is bellow 1%. It turns out that pure isopropyl myristate (90%) and PEG 400
are good solvents to form 1% solution, as they dissolve 10 mg/ml. PEG 400 was found to be more
advantageous solvent compared with PEG 300 and PEG 600, which respectively dissolved 5 mg/ml.
Generally water decreases solubility in organic solvents. It was proved that content of 20% of water in
-38-
solvent decreased solubility of TOL in isopropyl myristate below 1 mg/ml. If PEG concentration is
decreased to 80% dissolution fails even to 1 mg/ml. Only in case of isopropanol solubility decreased
gradually with increase of amount of water in the solvent. Content of 30% of water in isopropanol
decreased solubility almost 10 times to 1.1 mg/ml. When water was used as a solvent for surface tension
agents SDS (5%) and TW (5%) it was impossible to dissolve TOL even 1 mg per 1 ml (0.1%).
Table 7. Solubility of TOL in organic solvents at room temperature ( - solution). Solvent (% v/v)
ET IPM IPP CHLConcentration 95 90 70 90 80 100 90 80 70 50 100
20 mg/ml - - - - - - - - - - 10 mg/ml - - - - - - - - - 5 mg/ml - - - - - - - - -
3,33 mg/ml - - - - - - 2,5 mg/ml - - - - -
2 mg/ml - - - - - 1,67 mg/ml - - - -
1,43 mg/ml - - - - 1,25 mg/ml - - - -
1,11 mg/ml - - - 1,0 mg/ml - - -
Table 8. Solubility in organic solvents at room temperature ( - solution). Solvent (% v/v)
SDS TW PEG300 PEG400 PEG600 PGConcentration 5* 5* 100 90 80 100 90 80 100 90 80 100
20 mg/ml - - - - - - - - - - - - 10 mg/ml - - - - - - - - - - - 5 mg/ml - - - - - - - - 3,33 mg/ml - - - - - - 2,5 mg/ml - - - - - - 2 mg/ml - - - - - - 1,67 mg/ml - - - - - - 1,43 mg/ml - - - - - - 1,25 mg/ml - - - - - - 1,11 mg/ml - - - - - - 1,0 mg/ml - - - - - -
*concentrations are shown in % w/w
-39-
Organogels – preparation and properties
Although the dissolution results were not promising to reach 1% solubility of TOL in organogel
bases, an attempt to formulate a gel in a saturated state was made. On the basis of solubility results a
mixture of PEG 400 and ethanol was chosen for preparation of organogels. PEG 400 was also replaced
with propylene glycol (PG) for comparison, however recrystalization occurred in gels with PG just after
the heating was turned off. No recrystalization was observed in gels with PEG 400. However, undissolved
substance was seen under the microscope. Microscope pictures of prepared gels are shown in figure 12.
A
B C
Fig 12. Microscope pictures of organogels: A – OG1 (3% HPC + PEG), B – OG2 (5% HPC + PEG), C – OG3 (5% HPC + PG)
TOL solubility in oils
The alternative for a highly lipophilic substance to form a stable solution is to use lipophilic
solvents. In present study solubility was determined in vegetable oils: rape, olive, jojoba, Miglyol,
flaxseed, and castor. A standard procedure is to dissolve the substance in oils at 60-80oC, so it was
necessary to determine solubility after cooling the oil. Vegetable oils are colored and viscous, therefore
-40-
solubility determination was performed by microscope observation. For the proper crystal evaluation
while observing samples under the microscope, a suspension of TOL in water was prepared. Crystal size
was measured and was found to be in range of 5.6 and 21.6 μm. Microscope picture of TOL suspension in
water is shown in figure 13. TOL was found to be micronized because most particles were less than 10 μm
and just several particles reached the size of 22 μm.
Fig 13. TOL suspension in water (light microscope)
Visually when added to heated olive and rape oils TOL dissolved very quickly without mixing up
to 10% concentration, while in sunflower oil TOL dissolution took about 10 minutes after 3%
concentration was reached. While observing samples with 1% (w/w) TOL some crystals were seen in all
three oils. Crystal size was found to be around 10 μm, therefore it was assumed to be undissolved
substance. While observing samples with higher concentration of TOL it was found that recrystalization
starts to occur in olive oil after addition of 2% TOL, whereas in sunflower and rape oils TOL recrystalizes
after addition of 3% TOL. Also it was observed that recrystalization occurs in forming needles which tend
to grow in groups. TOL solutions in rape, flaxseed and Miglyol oils were also prepared. While observing
1% TOL solutions under the microscope undissolved substance was observed. Less and smaller crystals
were found in flaxseed and rape oils, if compared with Miglyol oil. It was expected that TOL should
easily dissolve in jojoba oil, because according to US Patent No. 4810498 [10] even a concentration of
50% can be reached with heating and agitation. However performed studies revealed that it is not possible
to dissolve TOL in jojoba oil even at 1% (w/w) concentration. Recrystalization was observed immediately
after placing 1% TOL solution in jojoba oil on the microscope slide. Examples of 1% TOL solutions in
oils are shown in figure 14. Crystal size was measured in sunflower, rape, olive and jojoba oils and is
summarized in table 9.
Fig 14. Microscope pictures of 1% TOL solution in oils
Table 9. TOL crystal size range in oils. Oil Crystal size range [μm]
TOL concentration 1% 2% 3% 4%
Sunflower 1.7 – 13.2 4.0 – 23.1 5.1 – 36.9 6.3 – 50.1
Rape 1.1 – 10.1 7.6 – 20.4 14.2 – 60.6 18.3 – 85.9
Olive 2.8 – 11.0 30.2 – 196.1 37.9 – 322.2 -
Jojoba 44.1 – 172.2 79.3 – 440.1 - -
Microscope slides and Petri plates with TOL solutions in oils were stored for a period of one
week. After 24 hours recrystalization of TOL was observed in 1% rape oil by microscope observation,
while other oils remained unchanged. After 7 days recrystalization was visible on all microscope slides
and Petri plates – they were covered in white crystals. Performed solubility studies revealed that it is not
possible to dissolve 1% (w/w) TOL in the examined oils. Crystals are seen in all oils at concentration of
1%. They are small but needles will start to grow as it was observed after 7 days.
1% olive oil 1% sunflower oil 1% rape oil
1% Miglyol oil 1% jojoba oil 1% castor oil
-42-
Viscous environment might sometimes slow or stabilize the recrystalization. Because of the aim of
the present study oleogels were prepared according to point 3.1 (methodology). Olive, Miglyol and jojoba
oils were chosen for oleogel preparations. Microscope observation revealed that less crystals were seen in
olive and Miglyol gels, if compared with jojoba gel (Fig 15).
Jojoba gel (G5) A B
Miglyol gel (G4) A B
Olive gel (G4) A B
Fig 15. Microscope pictures of prepared oleogels (A – light microscope, B – polarization microscope)
-43-
Prepared oleogel formulations were stored in plastic containers at room temperature. A change in
color and consistency was also observed (Fig. 16). Olive gel (G3) became deeper in color. Small color
change was observed in jojoba gel (G5). Gels were also unstable in consistency – oils separated from the
gel and were visible on the surface.
A B
Fig 16. Color change of oleogels stored in plastic containers: A – Jojoba gel; B – Olive gel
During storage recrystalization was observed in jojoba gel (G5) after 3 months. Observation after
10 months revealed that recrystalization had occurred in all 3 formulations. Most crystals were seen in
jojoba gel (G5). It was observed that needles tend to form at places which had interaction with external
environment – crystals were seen around air bubbles or places with sharp edges, for example places where
mixing took place. Recrystalization in jojoba oil is shown in figure 17.
Fig 17. Crystals in jojoba gel after 10 months stored at room temperature
Consistency evaluation of gel formulations
Consistency is an essential feature of semisolid dosage forms. In modern technology it is
necessary to perform rheology tests to evaluate physical properties and repeatability of series. HPC (3%,
-44-
5%) in organogels and aerosil (10%) in oleogels were used as gelling agents. On the first stage of
development of gels rheological properties were determined according point 4.2 (methodology) and are
shown in table 10.
Formulation Shear rate [1/s]
Viscosity, η [Pas]
Area [Pa/s] Thixotropy [Pa/s]
10516 ± 300.0 233.9 ± 131.7
37460 ± 1195.1 1120 ± 904
32890 5912
61820 29320
62420 33080
Obtained results indicate that organogel viscosity increased with increasing the concentration of
gelling agent. The increase of HPC concentration from 3% (OG1) to 5% (OG2) increased viscosity and
thixotropy almost 5 times. Both organogels were semisolid when observed organolepticaly, despite
viscosity of 3% gel was low. Oleogel viscosity measurements show dependence on the oil used. Jojoba gel
(G5) had similar viscosity and thixotropy as Miglyol gel (G4), while olive gel (G3) was 3 times less
viscous and had significantly smaller value of thixotropy. Gel rheograms are shown in Fig 18.
-45-
Fig 18. Rheograms of gel formulations: A – organogels, B – oleogels
Organogel flow curves indicate a stable rheological structure and show typical pseudoplastic
behavior. Gel with 5% HPC was found to be more rigid and had some noticeable thixotropic properties in
the hysteresis loop. In case of oleogels rheograms indicate that the gels are plastic. In the area of lower
shear rate oleogels behaved as pseudo plastic – the force was necessary for gel to star flowing, while in
higher shear rates shear stress decreased.
Acceptability and clinical efficacy of semisolid formulations require them to possess optimal
properties, such as ease of removal from the container and spreadability on the substrate. Spreadability of
gels was determined according to point 4.1 (methodology). Spreadability tests revealed that oleogels were
easier to spread if compared with organogels. Oleogel and organogel spreadability results are shown in fig
19 and 20.
Weight [g]
Ar ea
[c m
G5 G4
G3 OG2
Weight [g]
Ar ea
[c m
Organogel spreadability decreased with increasing the amount of gelling agent: gel with 3% HPC
was easier to spread than gel with 5% of HPC. It was found that oleogel spreadability depends on the oil
used. Miglyol (G4) and jojoba (G5) gels had similar spreadability, respectively 7.07 and 7.54 cm2, while
olive gel (G3) was more spreadable (10.17 cm2). Spreadability results were in agreement with rheological
properties of gels: gels which are less viscous and thixotropic are more spreadable.
Macrogol ointments – preparation and properties
It was impossible to dissolve TOL in organic solvents and their mixture at 1% concentration.
According to solubility studies PEGs can be used as single solvents to form 1% TOL solution. Solubility
was checked in pure liquid PEGs (300, 400 and 600) and was found to be above 5 mg/ml. To achieve a
semisolid consistency liquid PEGs were mixed with melted solid PEGs (molecular weight above 1000).
Several macrogol ointments were prepared according to point 3.3 (methodology).
In the first stage of consistency evaluation of MO5 spreadability was measured. Obtained results
(Fig. 21) show dependence on the ratio of PEGs used as well as on their molecular weight. Increasing the
amount of PEG 400 and lowering the amount of PEG 1500 by 10%, resulted in higher (2 times)
spreadability (MO1 changed to MO5). When the molecular weight of solid PEG was lowered from 1500
to 1000 spreadability also increased twice (MO1 changed to MO2). Macrogol ointment prepared
according to Polish Pharmacopoeia was found to be the least spreadable. MO2, MO4 and MO5 had
similar spreadability values, while MO3 spreadability was notably higher. Same results were observed
while squeezing ointments from the tubes – ointments with higher spreadability values were easier to
squeeze out.
Weight [g]
A re
a [c
m 2]
During further ointment development rheological properties were measured according point 4.2
(methodology) and are shown in table 11.
Table 11. Rheological properties of macrogol ointments.
Formulation Shear rate [1/s]
Viscosity, η [Pas]
Area [Pa/s] Thixotropy [Pa/s]
62553 ± 2086 21020 ± 1210
16620 3533
6316 1385
2539 591
22918 ± 2392 7597 ± 2125
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On the basis of rheology results it can be stated that viscosity and thixotropy of macrogol
ointments depend on the molecular weight and ratio of polyethylene glycols used. The decrease by 10% of
PEG 1500 in the proportion with PEG 400 (MO1 change to MO5) led to decrease of about 3 times in
viscosity and thixotropy. The change of PEG 1500 to PEG 1000 (MO1 change to MO2) also drastically
decreased viscosity (4 times) and thixotropy (6 times). Rheology results were found to be in disagreement
with spreadability results. MO2, MO4 and MO5 had similar spreadability values, while viscosity and
thixotropy values are different. It is also notable that macrogol ointment spreadability does not depend on
their viscosity. MO4 was found to be the least viscous while MO3 spreadability was higher.
MO2, MO3 and MO4 formulations exhibited pseudoplastic behavior (viscosity decreased with
increasing shear rate), while force was necessary to induct the flowing in formulations MO1 and MO5.
Rheograms of macrogol ointments are shown in figure 22.
Fig. 22. Rheograms of macrogol ointments
Creams – preparation and properties
Currently creams have become popular in topical treatment of fungal diseases, also TOL in
Lithuania is available only in a form of cream (Scholl Athlete’s Foot). Therefore in the present study an
attempt was made to prepare a TOL suspension in o/w cream with a high amount of water. Creams were
prepared according to point 3.4 (methodology). On the first stage of development cream rheological
properties (table 12) were measured according point 4.2 (methodology).
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Formulation Shear rate [1/s]
Viscosity, η [Pas]
Area [Pa/s] Thixotropy [Pa/s]
12073 ± 686.5 3722 ± 725
4961 ± 256.8 763.9 ± 27.2
3856 1318
11560 2665
16410 4772
10980 2094
1801 535
22120 9800
3951 888
3587 1039
2651 724
4377 599
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Cream rheology results revealed that water content did not have direct influence on viscosity and
thixotropy values of examined creams. Changes in rheological parameters were observed due to changes
in oily phase, emulsifiers and their ratios.
Creams C7 and C8 had almost the same amount of water phase, respectively 65% and 64%,
however C8 was found to be the most viscous and C7 one of the least viscous creams examined.
Formulation C7 had less solid lipids, as well as different types and ratio of emulsifiers, what resulted in
major decrease of viscosity and thixotropy (20 times) if compared with formulation C8. Even the change
of the proportion of emulsifier used has a huge influence on rheological parameters. Increasing the amount
of emulsifiers (C3 changed to C6) resulted in higher values of viscosity and thixotropy. The increase of
viscosity was 4.5 times, however thixotropy increased by only 1.6 times. It can be observed that flow
curves (Fig. 23) depend on the emulsifier system while water content does not have a significant influence
on rheological properties of creams examined.
Fig. 23. Rheograms of C3, C6, C7 and C8
Replacing solid components (petrolatum and cetostearyl alcohol) with liquid ones (water,
isopropyl myristate, liquid paraffin) resulted in decrease of viscosity 3 times, while thixotropy decreased 5
times (C1 changed to C2). Small changes were observed when isopropyl myristate was added to the
composition (C4 changed to C5) on the expense of cetostearyl alcohol, glycerol (amounts lowered by 2%)
and silicon fluid (amount lowered by 1%). The increase in viscosity and thixotropy was the same – 2
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times. Flow curves of formulations C4 and C5 are shown in figure 24. These formulations behave
pseudoplastic and had a rigid structure with linear flow.
Fig. 24. Rheograms of C4 and C5
In creams (C9, C11, C12) with the same emulsifier system, the influence of liquid lipid type:
isopropyl myristate, Miglyol oil and liquid paraffin on rheological parameters was observed. The change
of isopropyl myristate (C9) to liquid paraffin (C11) did not have a significant affect on rheological
parameters – both viscosity and thixotropy increased slightly. Noticeable change in viscosity was
observed when isopropyl myristate was changed to Miglyol oil (C12), which is a semisynthetic oil and has
low viscosity. In all three formulations Span 80 and cetostearyl alcohol were used as water in oil
emulsifiers. In formulation C15 Span 80 was changed to Span 40, however practically no change was
observed in viscosity and thixotropy, even though HLB values of these emulsifiers are different,
respectively 4.3 and 6.7. In formulations C8, C9, C11, C12 and C15 semisolid hydrocarbons were
changed to triglycerides (Precirol) in order to avoid greasiness and to have better absorption after
application. Precirol melts easily when affected by temperature or force, as a result untypical flow curves
are obtained (Fig. 25).
Fig. 25. Rheograms of C9, C11, C12 and C15
Due to untypical flow curves, caused by Precirol, gravitational separation test was used to check
the stability of cream (C9) structure. An amount of 5 g was placed to glass tubes and they were
centrifuged at 4000 rpm for 20 min. As seen in figure 26 no phase separation was observed in all three
samples, therefore it was stated that cream structure is stable.
Fig. 26. C9 samples after gravitational separation test
During further cream development spreadability was measured (Fig. 27). It was observed that
the increase of the amount of water (C1 changed to C2) or changes in liquid ingredients of lipophilic
phase (C9 changed to C11 or C12) did not change spreadability significantly. Creams had an average
spreadability of 21.24 ± 1.3 cm2 after 1163.7 g weight was applied. The composition of creams doesn’t
have a notable affect on spreadability, since the outer phase is water.
-53-
0
5
10
15
20
25
A re
a [c
m 2]
`
Fig. 27. Spreadability of creams
On the next stage of development of creams mechanical properties were analyzed using a non
pharmacopeial method according point 4.3 (methodology). Obtained results are summarized in table 13.
Texture profile results were in agreement with rheology results. The minimum and maximum values of
cream texture profiles were exhibited by the formulations C1 (maximum) and C2 (minimum). It was
observed that values of texture of creams do not depend on the amount of water used, whereas C8 (64%
water) texture parameters were bigger than in C11 (70% water) and smaller than in C1 (50% water).
Table 13. Texture profile of creams.
Formulation Firmness [g]
Consistency [gs]
Index of Viscosity [gs]
C1 101.4 ± 4.8 1669.9 ± 22.9 -45.5 ± 1.0 -801.7 ± 75.5 C2 21.6 381.6 -11.9 -240.4 C8 86.5 1584.4 -43.7 -703.9 C9 55.5 ± 0.8 994.3 ± 29.3 -29.0 ± 1.4 -469.7 ± 17.4 C11 68.1 1234.6 -32.4 -489.3
Formulation C1 was changed to C2 by increasing the amount of water, liquid lipids and by
decreasing the amount of solid lipids. These changes resulted in drastic decrease in all texture parameters.
The change of isopropyl myristate to liquid paraffin (C9 changed to C11) resulted in slightly higher values
of firmness and consistency (increased approximately 1.2 times), while cohesiveness and index of
viscosity did not change. Texture profile analyzes showed good repeatability and can be a good indicator
for properties of semisolids.
Preliminary dissolution studies
On the next stage dissolution studies were performed according to point 9 (methodology).
Macrogol ointment (MO1) and a cream (C1) were chosen for preliminary dissolution studies. At first UV-
Vis method was used for quantative determination of TOL in dissolution samples according to point 10.1
(methodology). Calibration curve (see point 10.1) was obtained by analyzing standard solutions of TOL.
The absorption spectrum of standard samples (see point 10.1) was found to be good, as 2 peaks with
maximum at 222 and 258 nm were registered. It was observed that PEG 400 increases absorption (Fig.
28). Therefore, during further studies dissolution samples were diluted 10 times to minimize PEG 400
concentration to 5%.
Fig. 28. Absorption curves of standard solutions (5 µg/ml) in pure ethanol and in 5% PEG400 solution in ethanol
During analyzes of dissolution samples with UV-Vis spectrophotometer problems with detection
of the first peak with maximum at 224 nm occurred, because of some interference from the sample.
Therefore, quantative determination of TOL was performed with HPLC according to point 10.2
(methodology). The average dissolutions (obtained from formulations MO1 and C1) determined by UV-
Vis and HPLC are compared in figure 29.
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A B
Fig. 29. Comparison of HPLC and UV-Vis methods: A – dissolution profile of MO1, B – dissolution profile of C1
As seen in figure above, results obtained by UV-Vis were higher than obtained by HPLC.
Chromatograms (Fig. 30) obtained by HPLC analyzes indicate that TOL is fully separated from other
substances present in samples and no peak interference occur. Furthermore the second peak (tR=2.7)
shows that the samples are contaminated with other substances. This contamination results in higher
absorption while analyzing samples by UV-Vis method. Therefore, for further quantative analyzes of
dissolution samples it was necessary to use HPLC method.
Fig 30. Example of HPLC chromatogram of cream dissolution profile sample (60 min)
Tolnaftate
Contamination
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Drug assay in creams method development
During further studies a method for TOL assay in creams has been developed. Content analyzes
were performed according to point 8 (methodology). Methanol, ethanol and methylene chloride were used
for TOL extraction from creams. Samples were either centrifugated or filtrated in order to remove lipids
from the solution. Methylene chloride was evaporated after filtering. Then the tube was filled with
methanol which was also evaporated in order to completely remove methylene chloride. After that 1 ml of
methanol was added to the sample and it was analyzed with HPLC. The results of method validation of
extraction with methanol, ethanol and methylene chloride are shown in table 14.
Table 14. Results of TOL extraction from creams.
Methanol Ethanol Methylene chloride
Cream extraction 91.0 ± 1.7% 88.7 ± 3.0% 94.6% 97.8% 95.9 ± 12.5%
Control sample 107.9 ± 10.1% 107.4 ± 6.9% 99.6% ± 0.1 - -
Methylene chloride is a very strong solvent, however the procedure of TOL extraction with this
solvent is complicated and time consuming. Content found with methylene chloride was simmilar to
content determined with ethanol extraction. In this case ethanol is more avantageous, since the method is
simple. Methanol was found to extract the least TOL, (around 90%). Overall, ethanol extraction was
chosen for further studies, since 99.6% of TOL were found in the control sample. Centrifugation was
chosen for seperating lipids, since almost 100% TOL was found in the centrifugated sample.
Macrogol ointment (MO5) - stability
It was taken into consideration that stability studies should ensure physical and chemical stability
of the products. Stability studies were performed according to point 11 (methodology) by performing tests
required by Ph. Eu. (drug content analyses, homogenicity evaluation) as well as recomended ones
(consistency and viscosity measurments, dissolution studies).
During storage organoleptic evaluation of macrogol ointment (MO5) was performed. Macrogol
oinment stored in 40oC after one day storage was found to be in liquid state. This was caused by either
higher temperature or high humidity. However, when the ointment was taken to room temperature it
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regained semisolid consistency. Macrogol oinment stored in 4oC was found to be more rigid than the one
stored in room temperature, however when taken to room temperature it regained normal consistency. No
change in color and odor was observed for MO5 during all storage period.
On the first stage of stability testing a macrogol oinment (MO5) was observed with polarization
microscope. Microscope observation was performed immediately after preparation (t=0), as well as after 1
(t=1) and 2 (t=2) moths for samples stored in all conditions. For proper stability evaluation pictures were
taken of MO5 base and base with suspended TOL. Some shining reflections were seen in the base picture
(Fig. 31 A), however crystals can be distinguished (Fig. 31 B).
A B
Fig. 31. Microscope pictures: A – MO5 base; B – TOL suspension in MO5 base
As seen in figure 32 after preparation (t=0) TOL was completely dissolved in the base. Pictures
of t=1 and t=2 revealed that there was no recrystalization of TOL observed in ointments for the whole
study period independent from temperature. On the basis of microscope pictures it can be concluded that
MO5 was stable in all conditions for the whole period.
Crystal group
B.1 B.2 B.3
C.1 C.2 C.3
Fig. 32. Microscope pictures of MO5 for stability evaluation: A – t=0, B.1 – t=1/4, B.2 – t=1/25, B.3 – t=1/40, C.1 – t=2/4, C.2 – t=2/25, C.3 – t=2/40.
On the next stage of stability tests, consistency evaluation of MO5 was performed. Spreadability,
rheological properties and texture profile was studied immediately after the preparation and after storage
in different conditions. Spreadability studies were performed at t=0 and after two months (t=2) for MO5
stored in 4oC and 25oC. Results are shown in figure 33.
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0
2
4
6
8
10
12
14
Weight [g]
A re
a [c
t=2/4
t=2/25
Fig. 33. Spreadability of MO5 samples stored in different temperatures (n=1)
Spreadability studies performed for MO5 represent a stable structure. Spreadability was found to
be similar during time independent on the storing temperature. Spreadability of MO5 stored in 4oC was
the same like after the preparation. Ointment stored in room temperature (25oC) was found to be less
spreadable. However, the decrease in spreadability was insignificant and is not an indication of instability.
On further consistency analyses rheological properties of MO5 samples were measured.
Experiments were carried out for ointment stored in 25oC after 1 and 2 months, while for ointments stored
in 4oC after 2 months. Obtained results (table 15) show that there is a tendency of increase in viscosity for
samples stored in both temperatures, since viscosity after 2 months was found to be higher than after the
preparation. The increase of viscosity by 20% was found in ointment stored in 25oC after 1 month and did
not change after 2 months, while viscosity was found to increase by 30% after 2 months in ointment
stored at 4oC.
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Table 15. Rheological properties of MO5 samples stored in different temperatures (n=6). Time/Temperature
[month / oC] Shear rate
Area [Pa/s] Thixotropy [Pa/s]
1.7 494.4 ± 77.3 9.9 73.5 ± 12.5t=0 30.0 29.9 ± 2.3
22918 ± 2391.5 7597.7 ± 2124.8
1.7 571.9 ± 62.0 9.9 83.8 ± 3.2t=1/25 30.0 36.6 ± 2.4
27346 ± 1081.2 7263.7 ± 593.1
1.7 534.3 ± 65.2 9.9 81.6 ± 3.6t=2/25 30.0 32.4 ± 0.9
25696 ± 867.3 8208.8 ± 446.0
1.7 595.9 ± 76.9 9.9 105.4 ± 9.5t=2/4 30.0 37.9 ± 3.9
31393 ± 1704.8 10139.3 ± 1766.1
As seen in the obtained results and rheograms (Fig. 34), thixotropy and area under the curve also
tend to increase, however the increase in viscosity was more pronounced.
Fig. 34. Rheograms of MO5 samples stored in different temperatures
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Texture analysis of MO5 (table 16) does not indicate any changes, because parameters measured
in the beginning and after 2 moths did not change significantly. Probably this method is less sensitive for
detecting changes in the structure than rheological measurements.
Table 16. Texture profile of MO5 samples stored in different temperatures (n=3). Time/Temperature
[month / oC] Firmness
[g] Index of Viscosity
[gs] t=0 184.6 ± 5.3 2896.3 ± 147.2 -149.1 ± 8.4 -2951.7 ± 168.7 t=1-25* 185.9 2951.7 -153.7 -3222.4 t=2/25 189.5 ± 10.9 2729.2 ± 257.5 -148.8 ± 13.0 -2943.2 ± 368.3 t=2/4 178.7 ± 16.1 2632.4 ± 320.4 -138.8 ± 7.9 -2623.6 ± 171.1
*single control measurement
During further studies pH of MO5 was measured (table 17). In MO5 after preparation pH was
found to be 5.5, which is close to physiological pH value of the skin. During time the pH tended to lower
depending on temperature. In 25oC after 2 months the pH was found to be lower by 0.73 units. In 40oC the
decrease in pH was found to be more pronounced – it lowered by 1.55 units.
Table 17. pH of MO5 samples stored in different temperatures (n=3). Time/Temperature
[month / oC] pH
[average ± SD] t=0 5.47 ± 0.14 t=1/25 4.73 ± 0.03 t=1/40 4.15 ± 0.10 t=2/4 4.74 ± 0.06 t=2/25 4.71 ± 0.01 t=2/40 3.92 ± 0.01
Stability studies were continued by content analyses of MO5 (table 18) according to point 8
(methodology). Results show, that there were no changes of TOL content in MO5 samples stored in 4 and
25oC. However, less TOL was found in samples stored at 40oC. Macroscopic observation of MO5 sample
stored in 40oC revealed that the containers were not tightly sealed. Therefore, MO5 might have been not
homogenic because of melting or humidity, which might have caused the decrease in TOL content.
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Table 18. TOL content found in MO5 samples stored in different temperatures (n=6). Time/Temperature
[month / oC] Content [%]
t=0 87.5 ± 2.90 t=1/40 97.2 ± 6.36 t=2/4 84.2 ± 0.03 t=2/25 84.9 ± 0.04 t=2/40 77.3 ± 0.02
Dissolution studies are very important for stability evaluation of semisolids. There is no
pharmacopoeial method for these studies. Dissolution studies were carried out according point 9
(methodology). According to solubility results (see tables 7 and 8) ethanol and PEG 400 mixture (1:1) was
chosen as a receiving medium. Cellulose membranes were used, to prevent mixing of hydrophilic
components with the receiving medium. Dissolution studies (table 19, fig. 35) of macrogol ointment
(MO5) revealed that in ointment stored at 25oC the decrease in the release kinetic is high and time
dependent. The dependency during two month storage is linear, since after one month the release was
found to be 2 times smaller and it again decreased 2 times after two months. There was also observed that
the temperature had influence on that process: drug release after 2 months from the sample stored in 4oC
was found to be similar to amount released after one month from the sample stored in 25oC. Low
temperature slowed the dicrease of kinetic profile.
Table 19. Drug release from MO5 samples stored in different temperatures (n=6). Drug release, % [Average ± SD] Time/Temperature [month / oC]Time [min]
t=0 t=1/25 t=2/25 t=2/4 30 5.3 ± 1.9 3.3 ± 0.6 3.3 ± 0.3 3.5 ± 0.9 60 8.9 ± 1.9 5.7 ± 1.5 4.6 ± 0.4 5.8 ± 1.1
120 16.1 ± 2.6 8.8 ± 1.2 6.8 ± 0.8 9.8 ± 2.0 240 28.4 ± 4.5 17.0 ± 2.2 9.7 ± 1.3 17.3 ± 5.2 360 37.4 ± 5.1 21.9 ± 3.3 11.8 ± 1.5 25.6 ± 9.2
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0
5
10
15
20
25
30
35
40
45
0 30 60 90 120 150 180 210 240 270 300 330 360 390
Time [min]
TO L
re le
as ed
T=0
T=1/25
T=2/25
T=2/4
Fig. 35. Drug release from MO5 samples stored in different conditions
Macrogol ointment was found to be instable, because the kinetic profiles were changed in time in
both storage temperatures. A huge decrease in TOL released was observed just after one month storage in
25oC. Usually such changes are caused by drug recrystalization in the base. Because no recrystalization
was observed and there is no change in TOL content, the reason for instability probably is the interaction
between TOL and PEGs. This was also confirmed by the measurements of pH – during 2 month study it
tended to lower dependent on storage temperature. Macrogol ointments can also be physically instable,
because during two month storage increase of viscosity parameters was observed (around 20%). However,
the change is too small to cause such drastic changes in dissolution profile of TOL. Storage in lower
temperature did not allow achieving stable dissolution profiles for two month storage.
Cream (C9) - stability
During macroscopic observation of cream (C9) no changes in consistency were observed
depending on storage conditions. No change in color and odor was observed. However, after one month
samples stored in 25oC were found to be contaminated, since growth of microscopic fungi was visible on
the sides and bottom of the container.
On the next stage cream (C9) was observed with polarization and light microscopes.
Observations of C9 pictures at t=0 (Fig. 36) revealed that the cream was homogenic – TOL crystals were
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seen in all areas observed. Crystal size was measured and was found to be 7.0 ± 2.0 µm. Lipophilic phase
had the average size of 5.4 ± 1.2 µm.
A B
Fig. 36. Microscope pictures of C9 at t=0: A – light; B – polarization (example of crystals are shown in black circles)
Observation of microscope pictures of C9 taken at t=1 (Fig. 37) revealed that there were no
changes in creams stored in all conditions after one month (t=1). Pictures taken at t=2 (Fig. 38) indicate
that creams stored at 25oC and 40oC are not stable.