This article was downloaded by: [McGill University Library] On: 17 September 2012, At: 02:17 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Dispersion Science and Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ldis20 Formulation of Self-Nanoemulsifying Drug Delivery System for Telmisartan with Improved Dissolution and Oral Bioavailability Javed Ahmad a , Kanchan Kohli a , Showkat R. Mir b & Saima Amin a a Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Delhi, India b Department of Phytochemistry and Pharmacognosy, Faculty of Pharmacy, Hamdard University, Delhi, India Version of record first published: 27 Jun 2011. To cite this article: Javed Ahmad, Kanchan Kohli, Showkat R. Mir & Saima Amin (2011): Formulation of Self-Nanoemulsifying Drug Delivery System for Telmisartan with Improved Dissolution and Oral Bioavailability, Journal of Dispersion Science and Technology, 32:7, 958-968 To link to this article: http://dx.doi.org/10.1080/01932691.2010.488511 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
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This article was downloaded by: [McGill University Library]On: 17 September 2012, At: 02:17Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Journal of Dispersion Science and TechnologyPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/ldis20
Formulation of Self-Nanoemulsifying Drug DeliverySystem for Telmisartan with Improved Dissolution andOral BioavailabilityJaved Ahmad a , Kanchan Kohli a , Showkat R. Mir b & Saima Amin aa Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Delhi, Indiab Department of Phytochemistry and Pharmacognosy, Faculty of Pharmacy, HamdardUniversity, Delhi, India
Version of record first published: 27 Jun 2011.
To cite this article: Javed Ahmad, Kanchan Kohli, Showkat R. Mir & Saima Amin (2011): Formulation of Self-NanoemulsifyingDrug Delivery System for Telmisartan with Improved Dissolution and Oral Bioavailability, Journal of Dispersion Science andTechnology, 32:7, 958-968
To link to this article: http://dx.doi.org/10.1080/01932691.2010.488511
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
Formulation of Self-Nanoemulsifying Drug DeliverySystem for Telmisartan with Improved Dissolution andOral Bioavailability
Javed Ahmad,1 Kanchan Kohli,1 Showkat R. Mir,2 and Saima Amin11Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, Delhi, India2Department of Phytochemistry and Pharmacognosy, Faculty of Pharmacy, Hamdard University,Delhi, India
To improve the dissolution and oral absorption of poorly water soluble drug-telmisartan, aself-nanoemulsifying system (SNES) was developed, characterized, and its relative bioavailabilitywas compared to commercially available formulation. Safsol-218, Tween-20, and Transcutol Pwere chosen as oil, surfactants, and cosurfactants respectively as they show highest solubilityfor telmisartan. The solubility of drug was further improved by adding sodium hydroxide(0.67%). The droplet size of the optimized emulsion was also evaluated and was observed to bein nano range. The dissolution of drug was rapid in simulated gastric fluid (pH 1.2) as well asin simulated intestinal fluid (pH 6.8) and was found to be pH independent. The pharmacokineticparameters (AUC0!t�SD, Cmax, and Tmax) of optimized formulation of telmisartan after oraladministration as SNES were compared with oral tablet and API suspension of drug. The resultsshowed a 4.34-fold increase in oral bioavailability of drug in comparison to tablet. The resultsdemonstrate that SNES composed of Sefsol-218, Tween-20, and Transcutol P substantiallyenhanced the bioavailability of telmisartan. Further, it showed highly significant fall(p< 0.001) in mean blood pressure of hypertensive rats for 48 hours. Thus, our study providesa useful oral dosage form for telmisartan.
Keywords Bioavailability, droplet size, self-emulsifying nano drug delivery, Safsol-218,telmisartan, Transcutol P
1. INTRODUCTION
Oral drug delivery has been the most attractive route ofdrug administration for treatment of various ailments,however, it is impeded owing to physicochemical character-istics of drugs such as high lipophilicity, poor aqueoussolubility, frequent liver metabolism, high intra- andinter-subject variability, and lack of dose-dependentresponse. With advanced research in homolipids and het-erolipids as excipients, lipid-based formulations to improvethe oral bioavailability of poorly water soluble drugs havegained much attention. From the viewpoint of oral drugdelivery, lipids are studied as components of various oilyliquids and dispersions that are designed to increase solu-bility and bioavailability of drugs from class II and IV of
the biopharmaceutical drug classification system.[1] Themost popular approach is the incorporation of theactive lipophilic component into inert lipid vehicles suchas oils, to form microemulsions, nanoemulsions, self-nanoemulsifying systems (SNES), and liposomes. The mostrecent approach is SNES, which is used to develop an orallipid dosage form for poorly soluble drugs. It is an isotropicmixture of drug with oil, surfactant, and cosurfactant thathas the ability to form fine oil in water (o=w) nanoemulsionsupon gentle agitation following dilution with the aqueousphase.[2] Digestive motility of the stomach and intestine pro-vides the required agitation for self-emulsification.[3–6] AsSNES self-emulsifies in the stomach and presents the drugin small droplets of oil, it improves drug dissolution by pro-viding a large interfacial area for partitioning of the drugbetween the oil and gastrointestinal (GI) fluid besidesimproving stability.[7,8] For drugs posing dissolution ratelimited absorption, SNES promises not only the enhance-ment in the rate and the extent of drug absorption but thereproducibility of the plasma concentration profile.[9–11]
Telmisartan is a potent, long-lasting, nonpeptide antagonistof the angiotensin II type-1 (AT1) receptor that is indicatedfor the treatment of essential hypertension. It selectively
Received 31 March 2010; accepted 8 April 2010.The authors are very grateful to Gattefosse (Saint Priest,
Cedex France) for providing the free gift samples of surfactants.J. A. acknowledges University Grants Commission for providingfinancial assistance for the project.
Address correspondence to Saima Amin, Department ofPharmaceutics, Faculty of Pharmacy, Jamia Hamdard, NewDelhi 110062, India. E-mail: [email protected]
Journal of Dispersion Science and Technology, 32:958–968, 2011
Copyright # Taylor & Francis Group, LLC
ISSN: 0193-2691 print=1532-2351 online
DOI: 10.1080/01932691.2010.488511
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inhibits stimulation of the AT1 receptor without affectingother receptor systems involved in cardiovascular regu-lation.[12] It is highly lipophilic and practically insoluble inwater resulting in its slow or incomplete dissolution in gas-trointestinal tract (GIT) and lesser bioavailability.[13] Thedrug shows pH dependent water solubility behavior and,therefore, is practically insoluble in aqueous solution inthe pH range of 3 to 9.[14]
Telmisartan is one of the most important antihyperten-sive drugs available, but its poor bioavailability remains achallenge. In order for telmisartan to be absorbed, it mustbe soluble in the GI fluids. However, if the dissolution rateis slow, there is a chance that an orally administered dosewill pass through the GIT without being absorbed. SNESmay present a solution for this problem through improvingthe rate of telmisartan dissolution. Therefore, the currentstudy is aimed at developing and evaluating an optimalstable self-nanoemulsifying formulation of telmisartanusing minimum surfactant concentration so that nano-sized droplets could be formed spontaneously in GIT,which remain stable on dilution by the GI fluids in orderto increase its bioavailability.
2. MATERIALS AND METHODS
2.1. Chemicals and Reagents
Telmisartan and Safsol-218 (PGMCE) were gifted byRanbaxy Research Laboratories (Gurgoan, India) andNikko Chemicals (Tokyo, Japan), respectively. Diethyleneglycol monoethyl ether (Transcutol P), caprylo caproylmacrogol-8-glyceride (Labrasol), polyglyceryl-6-dioleate(Plurol oleique), lauroglycol were gifted by Gattefosse(Saint Priest, Cedex France). Tween-20, Tween-80, PEG200, PEG 400, PG, isopropyl myristate (IPM), glyceroltriacetate (triacetin), oleic acid, castor oil, and ethanol(HPLC grade) were purchased from Merck (Schuchardh,Hokenbrunn, Germany). Acetonitrile and buffer were pur-chased from Fischer Scientific Co. (India). All chemicalswere of analytical grade.
2.2. Operating Conditions for HPLC Analysis
HPLC system (Shimadzu LC-10A VP) along withLichrospher C18 column (4.6mm� 250mm, 5 mm) wasused. The mobile phase was pumped at a flow rate of1ml �min�1 and the ultraviolet (UV) detector was set at295 nm. The mobile phase was acetonitrile and potassiumdihydrogen phosphate buffer pH 3.4 (60:40 v=v) and therun time was 10 minutes. The software used in thissystem was Class VP, Version 5.032.
2.3. Solubility Study
The solubility of telmisartan in various oils, surfactants,and cosurfactants was determined by adding an excessamount of drug in 2ml of various oils, surfactants, and
cosurfactants selected for screening of components andthese were mixed using a vortex mixer. The vials werethen kept at 25� 1.0�C in an isothermal shaker (NirmalInternational, Delhi, India) for 72 hours to reach equilib-rium. The equilibrated samples were removed from theshaker and centrifuged at 3000 rpm for 15 minutes. Thesupernatant was taken and filtered through a 0.45 mmmembrane filter. The solubility of telmisartan was determ-ined in oils, surfactants, and cosurfactants using HPLC at295 nm.
2.4. Pseudo Ternary Phase Diagram Study forFormulation Selection
To construct the phase diagram for optimization of thesurfactants and cosurfactants, oil and Smix (surfactant-cosurfactant, 1:1) were mixed well in different ratios, as1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The phase dia-gram was developed by the aqueous titration method usingdistilled water in the aqueous phase.
For determination of existence of zone of nanoemulsion,pseudoternary phase diagrams were constructed using theaqueous titration method. To construct pseudoternaryphase diagrams, the oil phase (Sefsol-218 in ethanolicmicroenvironment, 2:1) was mixed with the surfactant:co-surfactant phase (Tween-20 and Transcutol P, respectively)and the ratios of Smix used for titration was 1:0, 1:1, 1:2,1:3, 1:4, 2:1, 3:1, and 4:1. The mixture was titrated with dis-tilled water until it turned turbid. The pseudo ternary phasediagrams were constructed by plotting the water phase, oilphase, and surfactant: cosurfactant (Smix) phase used inthe experiment. From phase diagrams, oil, and Smix con-centrations were selected depending on the solubility ofthe intended dose of drug (20mg) in the oil at minimumSmix concentration.
2.5. Thermodynamic Stability Studies
From each phase diagram constructed, different formu-lations were selected from nanoemulsion region of pseudoternary phase diagram. Selected formulations were sub-jected to different thermodynamic stability tests.
2.5.1. Heating Cooling Cycle
Six cycles between refrigerator temperatures (4�C) and45�C, with storage at each temperature for not less than48 hours were studied. Those formulations, which werestable at these temperatures, were subjected to centrifuga-tion test.
2.5.2. Centrifugation
Passed formulations were centrifuged at 3500 rpm for 30minutes. The formulations which did not show any phaseseparation were selected for the freeze thaw stress test.
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2.5.3. Freeze Thaw Cycle
Three freeze thaw cycles between �21�C and þ25�Cwith storage at each temperature for not less than 48 hourswere studied for the formulations. Formulations thatpassed these thermodynamic stress tests were further sub-jected to dispersibility test for assessing the efficiency ofself-emulsification and=or robustness to dilution in GIT.
2.5.4. Dispersibility Test
The spontaneity of self-emulsification of oral nanoemul-sion was assessed using a standard USP XXII dissolutionapparatus 2.[15,16] Each formulation containing a dose ofdrug (20mg) was added dropwise to 200mL of either pur-ified water or 0.1N HCl at 37� 0.5�C. A standard stainlesssteel dissolution paddle rotating at 50 rpm provided gentleagitation.
The in vitro performance of the formulations was visu-ally assessed using the grading system. Grade A formula-tions rapidly form nanoemulsions, within 1 minute andare clear or bluish in appearance. Grade B formulationsare also rapidly formed but are a slightly less clear emul-sion with a bluish-white appearance. Grade C formulationsform fine milky emulsions that are formed within 2 min-utes. Grade D formulations are dull, grayish-white emul-sions having slightly oily appearance that is slow toemulsify (longer than 2 minutes are required). Grade E for-mulations exhibit either poor or minimal emulsificationwith large oil globules present on the surface. Formulationsthat passed the thermodynamic stability and dispersibilitytest with Grade A in purified water as well 0.1N HCl wereselected and diluted nanoemulsions were stored for 6 hoursand observed for any sign of phase separation or drugprecipitation.
2.6. Evaluation of Telmisartan Loaded SNES
Optimized SNES were evaluated for globule surfacemorphology, droplet size, zeta potential, viscosity, andin vitro drug release.
2.6.1. Transmission Electron Microscopy
Morphology and structure of the SNES were studiedusing transmission electron microscopy (TEM; Morgagni268D SEI, USA) operating at 200KV and of a 0.18 nmpoint-to-point resolution. Combination of bright field ima-ging at increasing magnification and of diffraction modeswas used to reveal the form and size of the globule.In order to perform TEM, the SNES was diluted withwater (1:100 v=v). A drop of diluted sample was thendirectly deposited on the holey film grid and observed afterdrying.
2.6.2. Droplet Size Analysis
The droplet size distribution of the diluted formulationswas determined in triplicate by photon correlation
spectroscopy using a Zetasizer (1000 HS, Malvern Instru-ments, UK). Light scattering was monitored at 25�C at a90� angle. All selected formulations were diluted with water(1:250 v=v) and mixed for one minute before testing.
2.6.3. Zeta Potential
The zeta potential of the diluted formulations was mea-sured using a Malvern Zetasizer (Nano ZS-90, Malvern,UK). The formulations were diluted with distilled water(1:250 v=v) and mixed for one minute using vertex mixer.Zeta potential of each formulation was determined intriplicate.
2.6.4. In Vitro Drug Release Study
In vitro release test was performed as per USP XXIVmethod using 500ml of freshly prepared dissolutionmedium (SGF pH 1.2 and SIF pH 6.8). The apparatuswas set at 100 rpm and was maintained at 37� 0.5�C.The dissolution was carried out using dialysis bag (MWCO12,000 g=mole; Sigma Aldrich, USA). The dialysis bag waspretreated by soaking it in the dissolution medium for 24hours prior to commencement of each release experi-ment[17,18] SNES formulation containing 20mg of telmisar-tan was placed in dialysis bag containing 2ml of thedissolution medium securely tied with a thermo-resistantthread. 1ml sample was withdrawn at regular time inter-vals and aliquot amount of dissolution medium wasreplaced to maintain sink condition. The release of drugfrom SNES formulation was compared with the suspensionof aqueous suspension containing 20mg of drug. The sam-ples were analyzed for the drug content using HPLCmethod at 295 nm. Two way ANOVA test was used tocompare the dissolution profile of formulations with thatof pure drug suspension.
2.7. In Vivo Study
Animal study was performed according to a protocolsubmitted and approved by Institutional Animal EthicsCommittee, Hamdard University (173=CPCSEA). The ani-mals used for in vivo experiments were adult Wistar femalealbino rats (weighing 180–200 g) kept under standard lab-oratory conditions, at temperature 25� 2�C and relativehumidity 55� 5%. The animals were housed in poly-propylene cages, three per cage, with free access to stan-dard laboratory diet (Lipton feed, Mumbai, India) andwater. Animals were divided in four groups of six each.Group I served as control, Group II was given SNES for-mulation TM1, Group III received marketed tablet andGroup IV received aqueous suspension. The administereddose of drug was 1.8mg=kg body weight. The rats wereanesthetized using diethyl ether and blood samples(0.5ml) were withdrawn from the tail vein of rat at 0(pre-dose), 0.5, 1, 2, 4, 6, 8, 10, 12, 18, and 24 hours inmicro centrifuge tubes containing EDTA as anticoagulant.
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Tubes were stored at room temperature, 25� 2�C and rela-tive humidity (55� 5%) for 30 minutes. The blood samplewas then centrifuged at 10,000 rpm for 10 minutes. Theplasma was separated and stored at �21�C until druganalysis which was carried out using RP-HPLC method.The collected plasma was analyzed by drug precipitationmethod.
Pharmacokinetic parameters were calculated by usingnon-compartmental analysis also called as Model inde-pendent analysis. All pharmacokinetic parameters (tmax,Cmax, AUC0!t) were calculated individually for each sub-ject in the group and the values are expressed asmean� SD. The comparative in vivo bioavailability pro-files of developed formulation TM1, tablet suspensionand API suspension were also deduced. The relativebioavailability (F) was calculated using the followingequation:
F ¼ ðAUC test=AUC referenceÞ � 100
The results of the study were statistically analyzed usingGraph Pad Instat 3, using ANOVA followed by Turkey-Kramer multiple comparison test, Values at p< 0.001 wereconsidered significant.
2.8. Pharmacodynamic Activity of Formulation in Rats
The antihypertensive activity of SNES formulation(TM1) as oral dosage form was evaluated by measuringthe blood pressure (BP) of albino Wistar rats by tail-cuffmethod using the BP measurement equipment (BiopacSystems Inc, USA). The equipment used was of a non-invasive kind suitable to measure the BP of alert ratsafter warming their tail for 60 seconds at 30� 0.5�C. Thesoftware used in this system was Biopac Student LabBSL PRO.
Animals were divided into four groups of six each. Formeasuring the blood pressure of animals through tail cuffmethod, the animals were trained to stay in the restrainerin a nonaggressive manner. Group I served as controland received no treatment during the entire study, althoughthe BP was constantly monitored. Groups II served as thetoxic control group; Group III served as the treatmentgroup; and Group IV served as the placebo control group.Groups II, III, and IV received the subcutaneous injectionof methyl prednisolone suspension 20mg=Kg bodyweightdose for two weeks to induce hypertension.[19] Rats withBP 140� 5mm Hg were treated as hypertensive and wereincluded for further study. After induction of hypertensionGroup III and IV were treated with SNES formulationTM1 and placebo formulation respectively while GroupII served as toxic control. The results of the study werestatistically analyzed using Graph Pad Instat 3, usingANOVA followed by Turkey-Kramer multiple comparisontest. Values at p< 0.001 were considered significant.
3. RESULTS AND DISCUSSION
3.1. Screening of Oils and Surfactants
To develop a SNES for oral delivery of poorly solubleand low bioavailable drugs such as telmisartan, the phar-maceutically acceptable oil, surfactant, and cosurfactantmust be chosen. The surfactants used in SNES formula-tions are known to improve the bioavailability by variousmechanisms.[4] Also, the addition of cosurfactant is knownto increase the extent of nanoemulsification region inpseudo ternary phase diagrams. For SNES, it is imperativethat the drug should be in its dissolved state, as it has beenshown that a greater concentration of the drug in dissolvedstate leads to high concentration gradient, which acts as adriving force for the permeation of drug through the GITmucosa leading to better bioavailability. For formulationpurposes, the solubility of telmisartan in oils was studied.Oils from different categories such as long chain triglycer-ide, medium chain triglyceride, as well as synthetic mono-glycerides were selected. The drug loading capability wasthe main factor when screening of oil phase was carriedout. The results of solubility of telmisartan in different oilsare shown in Table 1. Telmisartan showed high solubilityin Safsol-218 followed by Safsol-218-oleic acid (1:1) mix-ture. The solubility of telmisartan in Safsol-218 was foundto be 15� 0.071mg=ml, which is not sufficient to developunit size SNES formulation; therefore, to enhance itssolubility, 0.67% w=v NaOH as alkanizer was added. Thesolubility of drug then increased proportionately. Themaximum fields of self-emulsification were observed whenSafsol in ethanolic environment in 2:1 ratio with 0.67%w=v NaOH as alkanizer was used. The greater solubilityis because the drug is a weak acid with three distinct pKa
values, that is, 3.5, 4.1, and 6.0 suggesting the drug ioniza-tion at corresponding pH values ensuring dissolutionthroughout the GIT. Such increased solubility of drugs inethanolic microenvironment with alkali=acid has beenreported earlier.[10,20] Therefore, mixture of safsol in etha-nolic microenvironment in the ratio 2:1 was selected asoil phase.
Further, in pharmaceutical dosage form designing,mostly non-ionic surfactants are favored owing to theirstability, compatibility, nontoxicity, and pH independency.In the present study, different surfactants were screenedand drug showed highest solubility in Tween-20 andTween-80 (18.0� 0.092mg=ml and 15.5� 0.075mg=ml,respectively; Table 2). Among various cosurfactantsscreened for solubility, the drug showed higher solubilityin Transcutol P and Plurol oleique (11.5� 0.055mg=mland 10.9� 0.054mg=ml, respectively). Since there was aslight difference in solubility in surfactants and cosurfac-tants, phase diagrams were constructed to optimize thesurfactant and cosurfactant. Oil, surfactants, and cosurfac-tants were tested in four different combinations for phasestudies (results not shown) and on its basis, Tween-20and Transcutol P were selected as surfactants and cosurfac-tants, respectively.
3.2. Pseudoternary Phase Diagram Study
Surfactant and cosurfactant get preferentially adsorbedat the interface, reduce the interfacial energy as well as pro-vide a mechanical barrier to coalescence. The decrease inthe free energy required for the emulsion formation conse-quently improves the thermodynamic stability of thenanoemulsion formulation.[21] Cosurfactants are beneficialto form nanoemulsion at a proper concentration range. Butthe excessive amount of cosurfactant causes system tobecome less stable, although high intrinsic solubility ofdrug is observed, but this lead to the globule size increasewith the expanding interfacial film.[22] Therefore, theselection of oil and surfactant and the mixing ratio of oil
to surfactant:cosurfactant plays an important role in theformation of the nanoemulsion. Pseudoternary phase dia-grams were constructed to identify the self-emulsifyingnanoemulsion regions and to optimize the concentrationof oil, surfactants, and cosurfactants, the phase diagramsof the system containing Safsol-218 in ethanolic microen-vironment, Tween-20, and Transcutol P are shown inFigures 1.1–1.8. Although it is considered that the largecontent of oil in the formulation will be beneficial to SNES,a proper size distribution is also an important factor forselecting a self-nanoemulsifying vehicle. Ideally the concen-tration of oil between 20 and 50% generates droplet sizeranging from 24 to 110 nm. In our study, 180–220 ml ofoil depicted a narrow droplet size distribution. These SNESform fine o=w nanoemulsions upon dilution with gastricfluid but the free energy required to form nanoemulsionis very low and the formation is thermodynamically spon-taneous.[23] The effect of surfactant–cosurfactant ratio onthe solubility of telmisartan was also investigated. Anexcess amount of telmisartan was added to each Smix (mix-ture of Tween-20 and Transcutol P) ranging from 1:4 to3:1. It was observed that when Tween-20 was used alone(Smix ratio 1:0, Figure 1.1), large nanoemulsion gel areawas obtained while small o=w nanoemulsion region wasfound towards aqueous rich apex. The addition of Trans-cutol P (Figure 1.2) led to greater penetration of the oilphase in the hydrophobic region of the surfactant mono-mers thereby further decreasing the interfacial tension,which leads to increase in the fluidity of the interface andincreasing the entropy of the system.[24,25] When cosurfac-tant concentration was doubled, Smix ratio 1:2 (Figure 1.3),the total area of nanoemulsions decreased as compared toSmix ratio 1:1. When further cosurfactant concentrationwas increased to 1:3 (Figure 1.4) and 1:4 (Figure 1.5), thenanoemulsion area decreased. In contrast, when surfactantconcentration was increased as compared to cosurfactant,Smix ratio 2:1 (Figure 1.6), the concentration of oil thatcould be solubilized was increased but the nanoemulsionregion decreased as compared to 1:1. A small nanoemul-sion gel area was also observed (not shown), which maybe due to increased concentration of surfactant. Whenfurther surfactant concentration was increased to 3:1(Figure 1.7) and 4:1 (Figure 1.8), nanoemulsion area inthe phase diagrams slowly decreased with increase innanoemulsion gel area. Increase in gel character of formu-lation leads to delay in self-emulsification performance offormulation. Thus, in the present study, the optimal ratiosof Tween-20-Transcutol P selected were 1:1, 1:2, 1:3, 2:1,and 3:1.
3.3. Preparation of SNES
It is well known that large amount of surfactants causeGI irritation.[24–26] Therefore, it is important to determinethe surfactant concentration properly and to use minimum
TABLE 2Drug solubility in different surfactant and cosurfactant
concentration in the formulation. From each phase dia-gram constructed, different formulations were selectedfrom nanoemulsion region so that drug could be incorpor-ated into the oil phase and oil concentration should be suchthat it should dissolve single dose of the telmisartan, that is,20mg easily. Based on our results, a three componentSNES formulations were established containing 18–22%
Safsol-218 in ethanolic microenvironment as oil, 34–45%of surfactant and cosurfactants in various ratios. The com-positions of the optimized formulations (TM1–TM5) aregiven in Table 3.
Different compositions of SNES were subjected to ther-modynamic stability studies to ascertain that the preparedformulations were stable when subjected to freeze thaw
FIG. 1. Pseudoternary phase diagrams of the formulations composed of various proportions of Tween 20 and Transcutol P: 1.1 Tween 20: Trans-
cutol P (1:0); 1.2 Tween 20: Transcutol P (1:1); 1.3 Tween 20: Transcutol P (1:2); 1.4 Tween 20: Transcutol P (1:3); 1.5 Tween 20: Transcutol P (1:4);
1.6 Tween 20: Transcutol P (2:1); 1.7 Tween 20: Transcutol P (3:1); 1.8 Tween 20: Transcutol P (4:1). (Color figure available online.)
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cycle, centrifugation study, and heating cooling cycle. For-mulations that were stable and regained their original formafter freeze thaw cycle and did not show any phase separ-ation, turbidity, and change in color or drug precipitation,were then evaluated for dispersibility test or robustness todilution to see the self-emulsification potential and visualclarity after infinite dilution. The formulations TM1 toTM5 passed the dispersibility test with Grade A in distilledwater as well in 0.1N HCl, which indicates that these for-mulations readily form clear nanoemulsions upon infinitedilutions with simulated gastric fluid.[27]
3.4. Morphological Characterization
The drug loaded SNES turn to nanoemulsion whendiluted with aqueous phase. The droplet sizes are thenmeasured using TEM, as it is capable of point-to-point resol-ution. The nanoemulsion appears dark with bright surround-ings, thus, a positive image is seen using TEM. The globulesize of formulations TM1 to TM5 was observed to be spheri-cal. The TEM picture of TM1 is shown in Figure 2. Thenanoemulsions droplets were observed to be spherical.
3.5. Droplet Size Analysis
Droplet size analysis is a critical factor to evaluate aself-emulsifying nanoemulsion. Droplet size is known to
affect the drug absorption as studied by various research-ers. The smaller the droplet size, the larger is the interfacialsurface which leads to greater absorption. Also the stabilityof nanoemulsion is dependent on the particle size.[8] Thedroplet size distribution of diluted formulation TM1
(Figure 3) was smallest (43.33 nm) among all the tested for-mulations (Table 4). It was observed that increased concen-tration of oil and cosurfactant results in increase in dropletsize. The droplet sizes in case of TM2 and TM3 were foundto be more as compared to TM1 because formulations TM2
and TM3 contain higher cosurfactant. It was also foundthat cosurfactant (Transcutol P) did not play a vital roleas compared to the surfactant (Tween-20) for the emulsifi-cation of oil.
3.6. Zeta Potential
The emulsion stability is directly related to the magni-tude of the surface charge.[28,29] Generally it is observedthat when high electrical repulsive forces appear betweennanoemulsions droplets, then coalescence is preventedand more stable the tricomponent system becomes. Onthe contrary, a decrease in electrostatic repulsive forcescause phase separation.[2] In our study, zeta potential ofall the tested formulations was observed and is shown inTable 4. Formulation TM1 had higher zeta potential valuesthan other formulations that indicate that upon emulsifi-cation TM1 generates more stable emulsion than other for-mulations (Figure 4).
TABLE 3Composition of the tested SNES formulations
FIG. 2. TEM positive image of formulation (TM1) showing size of
some oil globules. FIG. 3. Droplet size distribution by volume of formulation TM1.
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3.7. In Vitro Drug Release Study
Conventional in vitro dissolution studies are not pre-ferred for self-emulsifying drug delivery system becausewhen such a three component system comes in contact withwater, then drug exists as a free molecule, as micelles or asnanoemulsion droplets. Under such conditions, it is desir-able to separate the isolated drug molecules from thetrapped drug molecules as micelles or as nanoemulsion.The use of dialysis bag in in vitro study provides consistentdrug dissolution throughout the dissolution study.[30] Thepercentage dissolution time profile of the five SNES formu-lations in SGF (pH 1.2) as well as SIF (pH 6.8) is shown inFigure 5 and 6. It was observed that from all formulationsthe release of drug is pH independent as was found to becomparable in both the dissolution medium. When the per-centage dissolution profile of all formulations was com-pared with API suspension of drug, there was higherdissolution of drug from SNES than the API suspensionin both SGF and SIF. It was observed from the dissolutionprofile that the drug release from formulation TM1 was sig-nificantly higher (p< 0.001). The lower release was observedwith TM2 and TM3 (63.32� 1.68 and 56.06� 1.2, respect-ively, in SGF).
The amount of drug dissolved in the aqueous phase atany time, t, is inversely proportional to the radius of thedroplets and the partition coefficient of drug Koil=aqueous
phase.[31] The measured partition coefficients of telmisartanin Safsol-218 are log oil=SGF¼ 2.94 and log oil= SIF¼ 3.14,that indicate that only 20–30% of drug is entrapped inthe oil phase while rest of the drug is present in the aqueousbiological fluid. The drugs partition coefficient inSafsol-218 is significantly lower than rest of the oilsscreened for the study, which indicates the superior dissol-ution profile of SNES formulations with Safsol-218.Further, the higher value of dissolution for TM1 formu-lation was because of markedly lower volume of oil takenas well as high concentration of surfactant and cosurfac-tant used to formulate the system. TM1 formulation alsofound to have highest zeta potential and smallest mean par-ticle size leading to a stable system. Therefore, the formu-lation TM1, having high drug release in both SGF andSIF (98.99� 0.59%, 97.89� 1.59%, respectively), smallestdroplet size (43.33 nm), and high zeta potential (�35.8mV)was selected for further study.
FIG. 5. Dissolution profile of drug at pH 1.2� SD, from (^) TM1,
(&) TM2, (�) TM3, (&) TM4, (~)TM5, and (.) API.
FIG. 6. Dissolution profile of drug at pH 6.8� SD, from (^) TM1,
(&) TM4, (�) TM3, (&) TM2, (~) TM5, and (-) API.FIG. 4. Zeta potential distribution of formulation TM1.
TABLE 4The mean particle size and zeta potential of diluted SNES
For poorly water soluble drugs like telmisartan, theabsorption is quite inadequate because of insufficient dis-solution of drug in aqueous medium i.e., drug shows dissol-ution dependent absorption. Once the self-emulsifyingsystem is formulated, complete emulsification of oil occursdue to surfactant and cosurfactants that delivers the drugin droplets of small size and complete dose of drug is avail-able in a dissolved form. The absorption of the drug then isdependent on transcellular or paracellular route. Usuallylipophilic drugs with low molecular weight are absorbedby transcellular route as it presents tight junctions asabsorption barrier. The surfactant–cosurfactant tends toreduce interfacial tension and enhance the transmembraneabsorption due to enhanced penetration of drug across thebiological membrane. Further, these SNES are present asdispersed particle in the gastrointestinal tract, which pro-vides larger surface area and rapid release of dissolved drugfrom SNES; thus, transport of drug through unstirredwater layer to the gastrointestinal membrane for absorp-tion is quite high. Moreover, these formulations are readilytaken up by lymphatic system thus further increasing thebioavailability of the drug with avoidance of first pass livermetabolism.[22]
Telmisartan plasma concentration values were assessedby the RP-HPLC, the method was sensitive and linear inconcentration range from 25 ng=ml to 4000 ng=ml withcoefficient of correlation 0.9963. The observed retentiontime for telmisartan was 5.05� 0.12 minutes. The pharma-cokinetic and plasma concentration time profiles of drug asAPI suspension, as tablet and SNES formulation TM1
were calculated using trapezoidal rule and are shown inTable 5 and Figure 7. Since SNES formulations are thecontrolled release formulations as the drug is released ina programmed manner from the oily phase of the nanoe-mulsion, therefore, the comparison of pharmacokineticbehavior of drug as SNES, as a tablet and as drug suspen-sion is required. The comparison is made on the basis ofplasma concentration after half an hour of administration(C1=2), and tmax. The mean C1=2 of formulation TM1 was 10
times higher than the tablet formulation with tmax value of2 ha. There was two-fold increases in C1=2 concentrationfrom marketed tablet formulation when compared withpure drug suspension. The mean AUC0!24� SD for for-mulation TM1 was calculated to be 24858.75� 1932.39,which showed a 4.3-fold increase in bioavailability of drugin comparison to marketed tablet of telmisartan. More-over, there was only a 1.8-fold increase in bioavailabilityof drug from marketed tablet formulation when comparedwith API suspension of drug.
Further, the results of mean AUC0!24� SD for formu-lation TM1 were when statistically analyzed in comparisonto marketed tablet, a significant increase (p< 0.001) inmean AUC0!24 was observed. This is because of the asso-ciated advantages of self-emulsifying drug delivery systemdiscussed earlier.
3.9. Pharmacodynamic Study
Hypertension was successfully induced in Group II, III,and IV rats by methyl prednisolone acetate administrationas highly significant difference (p< 0.001) was observed inBP compared to Group I. Glucocorticoids, like methyl pre-dnisolone, are known to cause systemic hypertension on
TABLE 5Relative pharmacokinetic parameters of different formulations containing telmisartan (n¼ 6)
aTime of peak concentration.bPeak of maximum concentration.cArea under the concentration time profile curve until last observation.dRelative bioavailability of formulations.eWith respect to API.
FIG. 7. Mean telmisartan plasma concentration-time profile� SD,
following oral administration of drug as (^) SNES, (&) tablet suspension,
and (*) API suspension to rats (n¼ 6).
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subcutaneous administration.[19] The results of the meanblood pressure with� SEM values for all the groups (i.e.,control, toxic control, toxic-treated-formulation TM1
(and placebo treated) are shown in Table 6. The plot ofmean BP of different groups is shown in Figure 8. On treat-ing Group III hypertensive rats with SNES formulationTM1, a significant fall (p< 0.001) in mean BP for 48 hourwas observed as compared to Group II while insignificantfall (p> 0.05) in mean BP was observed in Group IV ratsas compared to Group II as the former is the placebotreated group only.
4. CONCLUSIONS
Telmisartan was successfully formulated as self-emulsifying drug delivery system with Tween-20 andTranscutol P as surfactant and cosurfactant respectively.The formulation showed reproducible and reliable results
of in vitro dissolution study. The SNES of telmisartanshowed significant increase in oral bioavailability in ratsas well. However bioequivalence study of the formulationneeds to be tested on human volunteers to substantiateits market approval.
