Journal of Microencapsulation, August 2008; 25(5): 315–323 Development of an oral microemulsion formulation of alendronate: Effects of oil and co-surfactant type on phase behaviour FULYA KARAMUSTAFA & NEVIN C¸ ELEBI * Faculty of Pharmacy, Department of Pharmaceutical Technology, Gazi University, Ankara, Turkey (Received 11 May 2007; accepted 8 February 2008) Abstract This study aimed to prepare a water-in-oil microemulsion formulation of alendronate. Pseudo-ternary phase diagrams were constructed by using different oils and co-surfactants. The final formulation was decided to be prepared with Captex 200 Õ , lecithin, propylene glycol and bidistilled water. Rheological behaviour, phase stability and type of the microemulsion formulation were investigated by Brookfield viscosimeter, centrifugation test and dye method, consequently. Phase behaviour of the formulation was found to be Newtonian. No precipitation was observed in the stressed conditions and formulation was W/O. The physical characterization of the formulation (physical appearance, viscosity, refractive index, conductivity, density and turbidity) was investigated at 4 C and 25 C during 6 months while droplet size was investigated for 3 months. Droplet size of the formulation was between 224–280 nm while viscosity was between 89.9–99.5 mPa.S. The release of alendronate from the microemulsion formulation was examined by dialysis method and found to be 97.2% at the end of 7 h. None of the parameters except refractive index changed significantly during the determined periods. This study succeeded in preparing a stable microemulsion formulation of alendronate. Keywords: Alendronate, microemulsion, physical stability, phase diagrams, in vitro release Introduction In 1943, Hoar and Schulman first described water-in-oil microemulsions, which they referred to as transparent water-in-oil dispersions (Hoar and Schulman 1943). Microemulsions are thermodynamically stable, trans- parent, low viscosity and isotropic dispersions consist- ing of oil and water (Lawrence 1994). The dispersed phase of a microemulsion is enclosed by an interfacial film that consists of both surfactant and co-surfactant molecules arranged alternatively. A microemulsion can be one of three types: (1) oil-in-water (O/W), in which water is the continuous phase; (2) water-in-oil (W/O), in which oil is the continuous phase; and (3) bicontinuous, in which approximately equal volumes of water and oil exist (Tenjarla 1999). The tendency toward a W/O or an O/W microemulsion is dependent on the properties of the oil and the surfactant, the water-to-oil ratio and the temperature. W/O microemulsions are formed using surfactants which have a hydrophilic-lipophilic balance (HLB) in the range of 3–6 whilst O/W microemulsions are formed using surfactants which have a HLB value in the range of 8–18 (Leung and Shah 1989). W/O and O/W microemulsions have been shown to enhance the oral bioavailability of drugs. Drug delivery advantages offered by microemulsions includes improved drug solubilization and protection against enzymatic hydrolysis, as well as the potential for enhanced absorption largely due to the inclusion of absorption afforded by surfactant-induced mem- brane fluidity and thus permeability changes (Garcia- Celma et al. 1994). An increasing number of reports in the literature suggest that lipid-based microemulsions (W/O and O/W) can be used to enhance the oral bioavailability of drugs (Ritschel 1991, Constantinides et al. 1994, 1995, Kim et al. 2005), therefore it is important to understand the lipid digestion. Lipids are hydrolysed in the stomach and small intestine to the corresponding 2-monoglyceride (MG) and fatty acid (FA), absorbed into the enterocyte, re-esterified into triglyceride (TG) and packaged into Correspondence: Nevin C¸ elebi, Faculty ofPharmacy, Department of Pharmaceutical Technology, Gazi University, 06330- Etiler, Ankara, Turkey. Tel.: þ90 312 202 30 49. Fax: þ90 312 212 79 58. E-mail: [email protected]ISSN 0265–2048 print/ISSN 1464–5246 online ß 2008 Informa UK Ltd. DOI: 10.1080/02652040801977045 Journal of Microencapsulation Downloaded from informahealthcare.com by Aston University on 09/05/14 For personal use only.
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Journal of Microencapsulation, August 2008; 25(5): 315–323
Development of an oral microemulsion formulation of alendronate: Effects of oil
and co-surfactant type on phase behaviour
FULYA KARAMUSTAFA & NEVIN CELEBI*
Faculty of Pharmacy, Department of Pharmaceutical Technology, Gazi University, Ankara, Turkey
(Received 11 May 2007; accepted 8 February 2008)
AbstractThis study aimed to prepare a water-in-oil microemulsion formulation of alendronate. Pseudo-ternary phase diagrams wereconstructed by using different oils and co-surfactants. The final formulation was decided to be prepared with Captex 200�,lecithin, propylene glycol and bidistilled water. Rheological behaviour, phase stability and type of the microemulsionformulation were investigated by Brookfield viscosimeter, centrifugation test and dye method, consequently. Phase behaviourof the formulation was found to be Newtonian. No precipitation was observed in the stressed conditions and formulation wasW/O. The physical characterization of the formulation (physical appearance, viscosity, refractive index, conductivity, density andturbidity) was investigated at 4�C and 25�C during 6 months while droplet size was investigated for 3 months. Droplet size of theformulation was between 224–280 nm while viscosity was between 89.9–99.5mPa.S. The release of alendronate from themicroemulsion formulation was examined by dialysis method and found to be 97.2% at the end of 7 h. None of the parametersexcept refractive index changed significantly during the determined periods. This study succeeded in preparing a stablemicroemulsion formulation of alendronate.
Keywords: Alendronate, microemulsion, physical stability, phase diagrams, in vitro release
Introduction
In 1943, Hoar and Schulman first described water-in-oilmicroemulsions, which they referred to as transparentwater-in-oil dispersions (Hoar and Schulman 1943).Microemulsions are thermodynamically stable, trans-parent, low viscosity and isotropic dispersions consist-ing of oil and water (Lawrence 1994). The dispersedphase of a microemulsion is enclosed by an interfacialfilm that consists of both surfactant and co-surfactantmolecules arranged alternatively. A microemulsion canbe one of three types: (1) oil-in-water (O/W), in whichwater is the continuous phase; (2) water-in-oil (W/O), inwhich oil is the continuous phase; and (3) bicontinuous,in which approximately equal volumes of water and oilexist (Tenjarla 1999). The tendency toward a W/O or anO/W microemulsion is dependent on the properties ofthe oil and the surfactant, the water-to-oil ratio and thetemperature. W/O microemulsions are formed usingsurfactants which have a hydrophilic-lipophilic balance(HLB) in the range of �3–6 whilst O/W microemulsions
are formed using surfactants which have a HLB valuein the range of �8–18 (Leung and Shah 1989).W/O and O/W microemulsions have been shown to
enhance the oral bioavailability of drugs. Drug
delivery advantages offered by microemulsions includes
improved drug solubilization and protection against
enzymatic hydrolysis, as well as the potential for
enhanced absorption largely due to the inclusion
of absorption afforded by surfactant-induced mem-
brane fluidity and thus permeability changes (Garcia-
Celma et al. 1994). An increasing number of reports in
the literature suggest that lipid-based microemulsions
(W/O and O/W) can be used to enhance the oral
bioavailability of drugs (Ritschel 1991, Constantinides
et al. 1994, 1995, Kim et al. 2005), therefore it is
important to understand the lipid digestion.Lipids are hydrolysed in the stomach and small
intestine to the corresponding 2-monoglyceride (MG)and fatty acid (FA), absorbed into the enterocyte,re-esterified into triglyceride (TG) and packaged into
Correspondence: Nevin Celebi, Faculty of Pharmacy, Department of Pharmaceutical Technology, Gazi University, 06330- Etiler, Ankara, Turkey. Tel.: þ90 312 202
ISSN 0265–2048 print/ISSN 1464–5246 online � 2008 Informa UK Ltd.
DOI: 10.1080/02652040801977045
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intestinal lipoproteins. Intestinal lipoproteins, primarilychylomicrons and very low density lipoproteins, arefinally secreted into the mesenteric lymph from wherethey reach the systemic circulation via the thoraciclymph duct. Lingual and gastric lipase are responsiblefor the initiation of triglyceride hydrolysis forming thecorresponding diglyceride and fatty acid within thestomach. The liberation of these more amphiphilic lipiddigestion products, in combination with the shearproduced by antral contraction, facilitates formationof a crude emulsion which empties into the duodenum.The presense of lipid in the upper small intestinestimulates secretion of bile salts and biliary lipids fromthe gall bladder and pancreatic fluids from thepancreas. Biliary lipids such as phospholipids andcholesterol ester bind to the surface of the emulsifiedlipid, thereby improving colloid stability and reducingparticle size (Carey et al. 1983). The process of lipiddigestion is completed by the action of pancreatic lipaseand colipase which quantitatively produces two mole-cules of FA and the corresponding 2-MG from eachTG molecule (Borgstrom 1980).The phase behaviour of simple microemulsion
systems comprising oil, water and surfactant can bestudied with the aid of a ternary phase diagram inwhich each corner of the diagram represents 100%of that particular component. Almost in the caseof microemulsion in pharmaceutical applications, themicroemulsion will contain additional components suchas a co-surfactant and/or drug. In the case where fouror more components are investigated, pseudo-ternaryphase diagrams are used where a corner willtypically represent a binary mixture of two componentssuch as surfactant/co-surfactant, water/drug or oil/drug(Lawrence and Rees 2000). Phase diagrams are usefulin formulation studies as a means of delineatingthe area of existence of the microemulsion region(Bagwe et al. 2001).Surfactant concentration must be high enough to
stabilize the droplets. The type of the drug and theconditions of the target region is important for selectingthe surfactant (Bagwe et al. 2001). Non-ionic andzwitterionic surfactants are less toxic and are affectedfrom pH and ionic strength the least (Tenjarla 1999).Polyoxyethylene sorbitan esters (Tweens) are thesurfactants that are cheap and they are used mostly(Yaghmur et al. 2002). Non-ionic surfactants areconvenient for topical administration. Their applicationfor oral or parenteral delivery is limited (Tenjarla 1999).The biodegradation of many non-ionic surfactantscaused toxicity for chronic usage (Lawrence andRees 2000).Phospholipids are zwitterionic surfactants and show
perfect biocompatibility (Lawrence and Rees 2000).Lecithin is especially the most convenient surfactant forparanteral, oral and ocular administration (Pattarinoet al. 1993, Trotta et al. 1996). Lecithin, which isobtained from soybean or egg, is commercially
available and is composed of diachlyphosphotidylcholine as a major component (Zhang et al. 1996).Selecting the co-surfactant is an important parameter
for microemulsion formation (Bagwe et al. 2001).The role of the co-surfactant, usually a short-chainalcohol, is to increase the interfacial fluidity bypenetrating into the surfactant film and consequentlycreating a disordered film due to the void space amongsurfactant molecules (Leung and Shah 1989). However,the use of co-surfactant in microemulsions is notmandatory (Osborne et al. 1988). Microemulsionsneed co-surfactants, except the ones which are stabi-lized by non-ionic surfactants (Siebenbrodt and Kiepert1993). Stabilization can be obtained with a surfactant,but the mixture of surfactant–co-surfactant is necessaryfor constructing a mixed monolayer in the interfacialregion (Binks et al. 2003). In the absence of co-surfactant, lecithin forms reverse microemulsions overa very limited range of concentrations (Schurtenbergeret al. 1993). This is because of the lipophilic structureof lecithin (Cornell et al. 1986). In order to producea balanced lecithin microemulsion, a co-surfactant canbe used (Aboofazeli et al. 1994).Alendronate, a member of bisphosphonates, is used
in the prevention and treatment of post-menopausalosteoporosis (Bone et al. 2000), osteoporosis in men(Sharpe et al. 2001), coticosteroid-induced osteoporosis(Saag et al. 1998), treatment of Paget’s disease(Lin et al. 1993), primary hyperparatyroidism, malignhypercalcemia and metastatic bone diseases (Kirk andSpangler 1996).Alendronate is generally well-tolerated after short- or
long-term usage (Sharpe et al. 2001); but side effectssuch as oesophagitis and gastric damage were alsoreported (Graham et al. 1997).The bioavailability of alendronate is very low, less
than 1% (Porras et al. 1999). Oral bioavailability is0.64% in women when 5–70mg is taken after all nightfast and 2 h before breakfast. Its bioavailability isdiminished when the drug is taken with food, divalentcations like calcium (Gertz et al. 1995, Porras et al.1999), beverages other than water, with breakfastor within 2 h after breakfast (Gertz et al. 1995).Its bioavailability is increased with increasing gastricpH (Gertz et al. 1995, Porras et al. 1999). For reducingthe side effects and increasing the absorption, alendro-nate should be taken with water in the morning at least30min before breakfast and beverage. Patients shouldnot lie down for at least 30min and until the first foodof the day (Sharpe et al. 2001). For improving the oralabsorption of drugs, it is possible to formulate them indrug carrier systems. Microemulsion is a convenientway for drug delivery.In this regard, the development of a microemulsion
formulation of alendronate would have tremendousclinical importance as it would result not only inreducing the side effects and discomfort of drugadministration but also better patient compliance.
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There is no study to increase the bioavailability ofalendronate and reduce its side effects so the objectiveof this study is to develop a microemulsion formulationof alendonate for improving its oral bioavailability andreducing the side effects. For this purpose, two differentoils (Captex-200� and Captex-355�) and co-surfactants(absolute alcohol and propylene glycol) which arecapable of forming microemulsions were investigated.A W/O microemulsion was formulated in whichalendronate was trapped in the water phase. Thecharacterization of the prepared microemulsion wasinvestigated to prove that the formulation was stablefor the determined periods.
Materials and methods
Materials
Alendronate sodium trihydrate was kindly providedfrom Sanovel Pharmaceutical Company (Turkey).Captex-200� (C8/C10 diesters of propylene glycol fromcoconut oil) and Captex-355� (triglycerides from coco-nut oil) were kindly donated from Abitec Corporation(Japan). Phospholipon 90 NG� (Lecithin) was suppliedby Nattermann Phospholipid GmbH (Germany).Propylene glycol and absolute alcohol were suppliedby Sigma and Riedel-de Haen (Germany), respectively.
Methods
Formulation studies. To understand the effects ofexcipients on the formulation of a microemulsion,three different formulations including different oilsand co-surfactants were selected (Table I).Pseudoternary phase diagrams were established todetermine the microemulsion field. A titration techni-que was used for this purpose. The surfactant/co-surfactant (S/Co–S) ratios (Km) for all formulationswere decided to be 0.5, 1, 1.5 and 2. Lecithin wasdissolved in the co-surfactant (propylene glycol orabsolute alcohol) in the ultrasonic bath. Oil phase(Captex-200� or Captex-355�) was added to themixture and stirred with a magnetic stirrer. The mixturewas titrated with bidistilled water at 25� 0.5�C.Titration was stopped with the presence of a cloudysystem. The microemulsion region was identified as thearea in the phase diagram where clear and transparentformulations were obtained based on visual inspection.Formulation 1 was prepared containing alendronate,
but it was recognized that alendronate precipitated after
a while. The reason for the precipitation was thought tobe due to alcohol, because alendronate is known to beinsoluble in alcohol so the formulation which wouldbe examined for the stability experiments was decidedto be formulation 3.We tried to find the most convenient point for
preparing the formulation in the field obtained for theS/Co-S ratio of 1. The formulation was prepared fromthe point which is comprised of 46% Captex-200�, 25%lecithin, 24% propylene glycol and 6% bidistilledwater. The desired amount of drug was weighed outand then dissolved in the appropriate amount ofaqueous phase.
Rheology studies. The rheological behaviour of themicroemulsion formulation was determined by using arotating-spindle (spindle no: 40) Brookfield viscosi-meter. The rheology was evaluated by plotting shearstress vs. shear rate values obtained experimentally.
Phase separation and type of the microemulsionformulation. To understand if phase separationoccurs in stressed conditions, the formulation at roomtemperature was centrifuged at 1200 g for 5 h.To observe if the microemulsion is W/O or O/W, two
dyes were used, methylene blue and sudan III, whichare dissolved in water and oil phase, respectively.
Physical characterization of the microemulsionformulation. Physical stability of the prepared micro-emulsion formulation was evaluated by monitoringthe time- and temperature-dependent change in thephysical characteristics. For this purpose, physicalappearance, viscosity, droplet size, refractive index,conductivity, density and turbidity of the formulationwhich was kept at 4�C and 25�C were investigated. Themeasurements were performed every month during6 months, while droplet size of the formulation wasinvestigated for 3 months. All measurements wereperformed at room temperature.The physical appearance of the microemulsion
formulation was observed. The viscosity of theformulation was measured with a Brookfield viscosi-meter at 25� 0.1�C (spindle no: 40).Droplet size of the microemulsion formulation was
measured by using Zetasizer Nano ZS (Malvern). Thepredetermined viscosity of microemulsion formulationwas incorporated into the computer software whichcalculates the mean droplet size and polydispersity fromintensity and volume distribution. The results were
Table I. Compositions of various microemulsion formulations.
Formulation 1 Captex-200� Lecithin Absolute alcohol Bidistilled water
Formulation 2 Captex-355� Lecithin Absolute alcohol Bidistilled water
Formulation 3 Captex-200� Lecithin proplylene glycol Bidistilled water
Development of an oral microemulsion formulation of alendronate 317
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expressed as intensity distribution. Malvern systemmeasures the droplet size by relying on Brownianmovements with dynamic light scattering. Dropletsmove randomly in the liquid system. Small dropletsmove rapidly while bigger ones move slowly. The rateof them was used for determining the size of thedroplets.Refractive index, conductivity and density of the
formulation were determined using a Schimadzu�
refractometer, HI 9033 Henna Instruments and liquidpicnometer, respectively.The turbidity of the formulation was measured with a
Hach Model 2100A turbidimeter. During the measure-ments, 100 NTU (nephelometric turbidity unit) wasused as a blank for calibrating the turbidimeter.
In vitro release studies. Microemulsion formulationcontaining a certain amount of alendronate was settledto the donor chamber of the Franz diffusion apparatuswhich was separated with a membrane that is perme-able of molecules smaller than 12 000 Dalton.Bidistilled water was settled to the receptor chamberand stirred with a magnetic stirrer. Temperature wasmaintained at 37�C. Samples were withdrawn from thereceptor chamber at various time intervals during 7 h.The volume removed from the receptor chamber wasalways replaced with fresh pre-warmed water in orderto maintain the receptor fluid as constant. The sampleswere analysed for alendronate content spectrophoto-metrically at 262 nm. Alendronate was unable to bedetected due to the lack of a suitable UV chromophore.Thus, the derivization procedure described byDe Marco et al. (1989) was performed. The spectro-photometric method was validated from the points oflinearity, accuracy, repeatability, precision, selectivity,specificity, limit of detection and limit of quantification.
Analysis of data. Independent-sample t-test wasused for data analysis. Results were expressed as themean� SD, p5 0.05 was considered significant for allcomparisons.
Results and discussion
Formulation studies
Alendronate is a water-soluble drug and its side effectscan be observed when it is not taken with water in themorning at least 30min before breakfast and beverage(Sharpe et al. 2001). Preparing a W/O microemulsionformulation in which alendronate is in the aqueousphase was thought to be a way for handicapping its sideeffects. Oral bioavailability of alendronate which isreported to be too low (Porras et al. 1999) was alsothought to be enhanced with the prepared W/Omicroemulsion.Constructing the pseudo-ternary phase diagrams is
an important stage for developing a microemulsion
formulation (Bagwe et al. 2001). This sudy aimed toprepare a formulation for oral usage so it wasimportant to select the convenient excipients.Formulations with different excipients were studiedfor this purpose. The effects of different oils(Warisnoicharoen et al. 2000, Kawakami et al. 2002,Ke et al. 2005), surfactants and co-surfactants(Kawakami et al. 2002, Ke et al. 2005) on microemul-sion formation had been studied by many researchers.Using lecithin as a surfactant shows perfect biocom-
patibility (Lawrence and Rees 2000). There are manystudies with lecithin-based microemulsion formulationand characterization (Schurtenberger et al. 1993,Aboofazeli et al. 1994, Cilek et al. 2006). Based onthese data, it was decided to use lecithin as a surfactant.Short and medium chain alcohols except ethanol are
not convenient due to their toxicity. The stability of thesystem can be discomposed with the evaporation ofalcohol (Tenjarla 1999), which makes it of limited use.Propylene glycol is known to be used orally
(Kawakami et al. 2002). The formation and character-ization of phospholipid microemulsion and the effectsof different co-surfactants on the pseudo-ternary phasediagrams of water/lecithin/isopropyl myristate systemshad been studied (Aboofazeli et al. 1994). The improvedwater and oil solubilization in the presence of polyols(propylene glycol and glycerol) and short-chain alcohol(ethanol) in W/O and O/W food microemulsions hadbeen investigated (Yaghmur et al. 2002). According tothese data, it was decided to try absolute alcohol andpropylene glycol for the formulations. The aim was tosee the effect of co-surfactant type on microemulsionexistence field.The solubility and absorption of drugs can be
increased with polyglycolysed glycerides (Tenjarla1999). Modified and hydrolysed vegetable oils canalso be used in microemulsion formulations(Constantinides 1995). Medium chain glyceridesderived from coconut oil are attractive oils for oraladministration (Tenjarla 1999). Captex-200� andCaptex-355� are derivatives of coconut oil(Constantinides 1995) and they can be used orally(Patil et al. 2004). It was decided to use them for theformulations in order to see the effect of oil type on themicroemulsion existence field.When the microemulsion areas for formulations
1 and 2 were compared, it was obvious that largerareas were obtained with Captex-200� (Figures 1–4). Itcan be concluded that oil type can be a factor for themicroemulsion area. Based on these data, it was decidedto prepare a microemulsion using formulation 1.The largest area for formulation 1 was obtained withthe Km ratio of 1.5 (Figures 1 and 3). Weprepared a microemulsion containing alendronate butrecognized that alendronate precipitated after a while.The reason for the precipitation was thought to be dueto alcohol, because alendronate is known to beinsoluble in alcohol.
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Thus, it was decided to try propylene glycol as a
co-surfactant. The microemulsion areas obtained in the
presence of propylene glycol (Formulation 3) (Figures 5
and 6) were smaller when compared with the ones
containing alcohol (Figures 1 and 3). Constantinides
and Scalart (1997) had found that the field obtained
with Arlacel 186 (contains propylene glycol)/soybean
oil/Tween 80/water was smaller than the field of
soybean oil/Myverol 18-99/Tween 80/water. It was
thought that propylene glycol in Arlacel 186 had
made the field smaller.The largest area for formulation 3 was obtained with
the Km ratio of 1 (Figures 5 and 6). It was important to
select a convenient point for preparing the formulation.
Ritschel (1991) used the centre of gravity for preparing
their formulation. Selecting the centre of gravity can be
an advantage because that point represents the whole
field. Thus, we used centre of gravity for the final
formulation, which would be investigated from the
point of stability. However, the solubility of alendro-
nate limited us to use this point. It was important to
select a point which was not so near to the borders of
the microemulsion region, because approaching the
borders could easily spoil the stability of the formula-
tion on the occasion of small changes like temperature.
Thus, the microemulsion formulation was prepared
from the point as defined above.When a microemulsion was prepared according to
formulation 3, the precipitation of alendronate was not
observed.
Rheology studies
When shear stress is plotted vs. shear rate, it was
observed that microemulsion formulation showed
Newtonian flow behaviour (Figure 7). The rheological
behaviour of dilute systems such as a microemulsion is
generally known to be Newtonian (Cilek et al. 2006).
When the system shows Newtonian behaviour, it is
understood that droplets are small and spherical
(Kristis 1990).
Figure 3. Pseudo-ternary phase diagrams of microemulsion (Formulation 1) using a Km of 0.5, 1, 1.5, 2.
0
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50
60
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2
Figure 1. Relative areas of microemulsion (Formulation 1)existence field as a function of S/Co-S ratios.
0
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Figure 2. Relative areas of microemulsion (Formulation 2)existence field as a function of S/Co-S ratios.
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Phase separation and type of the microemulsionformulation
In the literature, microemulsions had been tested forstability by means of repeated centrifuging for 30min at13 000 rpm (Gasco et al. 1989) and at 1200 g for 5 h(Turkyilmaz et al. 1997, Cilek et al. 2006). Theformulation was investigated for 5 h at 1200 g and noprecipitation was observed in the stressed conditions.The type of the formulation was found to be W/O,
which is in accordance with the results found by Cileket al. (2006) and Turkyilmaz et al. (1997).
Physical characterization of the microemulsionformulation
It is important to characterize the physical propertiessuch as droplet size, turbidity (Turkyilmaz et al. 1997,Yetkin et al. 2004, Cilek et al. 2006), viscosity,density, refractive index (Kristis 1990, Constantinides
et al. 1994, Turkyilmaz et al. 1997, Yetkin et al. 2004,Cilek et al. 2006) and conductivity (Turkyilmaz et al.1997, Cilek et al. 2006) of the formulation. Theseproperties provide useful information for the stabilityof the microemulsions.Constantinides and Scalart (1997) evaluated the
shelf-life stability of microemulsions, both as a functionof time and storage temperature routinely by visualinspection of the samples initially on a daily and lateron a weekly basis. Stable systems had been identified asthose free of any physical change such as phaseseparation, flocculation and/or precipitation. We inves-tigated the physical appearance of the developedformulation. No change was observed at 25�C, but aturbidity was observed in the formulation which waskept at 4�C. When the formulation at 4�C was broughtto room temperature, the turbidity disappeared. It wasthought to be because of lecithin. The same results werefound by Cilek et al. (2006). Viscosity measurementscan indicate the presence of rod-like or worm-likereverse micelles (Angelico et al. 1998). Viscosity is animportant consideration in the design and stability ofemulsions and semisolid pharmaceutical dosage forms.The authors carried out the viscosity measurements for6 months (Figure 8) and found no significance betweenthe formulations at different temperatures (p4 0.05).It is important to determine the droplet size of
dispersed phase (Tenjarla 1999). Droplet size of themicroemulsions upon storage had also been deter-mined to assess microemulsion stability in terms ofdrastic changes in the mean droplet diameter due todroplet coalescence and/or aggregation (Constantinidesand Scalart 1997). The droplet size of the microemul-sion formulations were measured for 3 monthsand no significant difference was obtained betweendifferent temperatures (p4 0.05). Droplet size of themicroemulsion formulation during 3 months was in the
Figure 4. Peudo-ternary phase diagrams of microemulsion (Formulation 2) using a Km of 0.5, 1, 1.5, 2.
0
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0.5 1 1.5 2Rel
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Figure 5. Relative areas of microemulsion (Formulation 3)existence field as a function of S/Co-S ratios.
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range of 254–280 nm for 25�C while it was 224–251 nmfor 4�C. The droplet size results for 3 months are shownin Figure 9.On the other hand, in the laboratories, two different
microemulsion formulations were developed and thedroplet sizes were found to be 5 nm (Cilek et al. 2006)and 6–10 nm (Turkyilmaz et al. 1997).
The constancy of the refractive index is a sign of theconstant microemulsion structure (Cilek et al. 2006).This study investigated the refractive index of theformulations and found that there was a significantchange (p5 0.05) (Table II).Conductivity measurements provide a means of
determining whether a microemulsion is oil or watercontinuous as well as providing a means of monitoringpercolation or phase inversion phenomena (Mehta et al.1999). O/W microemulsions are highly conducting,whereas W/O are not (Constantinides 1995). The lowelectrical conductivity of the microemulsion formula-tion shows the oil structure of the external phase (Cileket al. 2006). Constantinides and Scalart (1997) pointedout that low conductance (5 1 mmhos cm�1) had beenemployed to verify that the microemulsions formedwere W/O type. The conductivity of the microemulsionswas measured and no significance was found atdifferent temperatures (Table II) (p4 0.05).
Figure 6. Pseudo-ternary phase diagrams of microemulsion (Formulation 3) using a Km of 0.5, 1, 1.5, 2.
Figure 9. The droplet size values of the microemulsion(Formulation 3) which was kept at 4�C and 25�C during3 months (M� SD, n¼ 3).
Figure 8. The viscosity values of the microemulsion(Formulation 3) which was kept at 4�C and 25�C during6 months (M� SD, n¼ 3).
0
50
100
150
200
250
300
0 50 100 150 200 250
Shear stress (mPa. S)
She
ar r
ate
(sn-
1)
Figure 7. Rheological diagram of the microemulsion(Formulation 3) at 25�C (M� SD, n¼ 3).
Development of an oral microemulsion formulation of alendronate 321
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The density change in the microemulsion is indicativeof the evaporation of the volatile compound in theformulation (Cilek et al. 2006). There was not a volatilecompound in the formulation, so no change wasexpected, as had been proven by the results (Table II)(p4 0.05).Turbidity experiments are important to estimate the
extent of clustering/growth (Fletcher and Morris 1995).The turbidity of the formulations (Table II) did notchange significantly during 6 months (p4 0.05).Constantinides and Scalart (1997) investigated the
viscosity, refractive index and conductance. They foundthat microemulsions with low HLB surfactantshad 5–30 nm droplet diameters. Low polydispersityindex (50.2) was indicative of monodispersed (homo-geneous) particles. These microemulsions could bestored without any precipitation and phase separationfor a few months at 4�C, 30�C and 40�C.
In vitro release studies
Drug release from the microemulsion is important tocharacterize the microemulsion formulation. It dependson several factors; oil–water partition coefficient, phasevolume ratio, droplet size of the dispersed phase,distribution of the drug in the various phases of thesystem, potential interaction between the excipients and
drug and the rate of drug diffusion in both phases of the
system (Tenjarla 1999). According to in vitro release
experiments performed by using Franz diffusion
apparatus, it was determined that 46.7%, 66.4% and
97.2% of alendronate was released during 1, 2 and 7 h,
respectively (Figure 10).
Conclusion
The results indicated that the physical stability of the
developed microemulsion formulation did not change
under different storage temperatures for the determined
periods. Thus, we succeeded in preparing a stable
microemulsion formulation for alendronate. This for-
mulation can help to improve the bioavailability of
alendronate for further studies.
Acknowledgements
This study was supported by grants from GaziUniversity (02/2004-29-Turkey). We want to thank
Sanovel Pharmaceutical Company (Turkey) for kindly
providing alendronate sodium trihydrate.
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