INTERNATIONAL JOURNAL OF PHARMACEUTICAL AND CHEMICAL SCIENCES

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1 Reserh Artile Frition nd Evlution of Atorvsttin Clium Loded Sustined Relese Mirospheres using O/W/O Doule Emulsion Solvent Evportion Tehnique Neelm Singh*, Tni Munjl, Sukhir Singh nd Sndeep Aror Chitkr College of Phrmy, Chitkr University, Chndigrh Ptil Ntionl Highwy (NH-64), Tehsil-Rjpur, Distt-Ptil , Punj, Indi. ABSTRACT Mirospheres re one of the multiprtiulte delivery systems nd re prepred to otin prolonged or ontrolled drug delivery, to improve iovilility or stility, reduing side effets nd improving ptient ompline. A doule emulsion solvent evportion method ws employed for the preprtion of Atorvsttin Clium Mirospheres using Eudrgit RL 100 nd Eudrgit RS 100 polymers. The influene of formultion ftors (polymer: drug rtio, emulsifier onentrtion, visosity of queous phse, stirring speed nd stirring time) on prtile size, morphology, enpsultion effiieny, drug loding, proess yield nd in vitro relese ehvior ws studied. The in-vitro performnes of mirospheres were evluted y reovery effiieny, prtile size nlysis, surfe topogrphy (using snning eletron mirosopy), drug-polymer omptiility (FTIR) nd drug relese studies. The o/w/o emulsion solvent evportion method ws suitle for the preprtion of mirospheres in the size rnge of ± 1.06 μm, the enpsultion effiieny ws ± 1.21% (w/w) nd the proess yield ws ± 1.24% (w/w). SEM reveled tht miroprtiles were smooth, spheril in shpe. FTIR, XRD studies showed no potentil hemil intertion etween the drug nd polymer used. In vitro relese studies reveled ontrolled relese of mirospheres suitle for perorl dministrtion. Drug relese from mirospheres followed Higuhi kinetis. Keywords: Solvent evportion method, Entrpment effiieny, Proess yield, Surfe topogrphy. INTRODUCTION Conventionl orl drug dministrtion does not usully provide rte-ontrolled relese or dosge in order to mintin dequte plsm onentrtion. Beuse of poor iovilility nd rther high first pss metolism, it is trget speifiity. In mny ses, onventionl neessry to develop sustined relese drug delivery provides shrp inrese of drug preprtion with extended linil effet 3-7. onentrtion t potentilly toxi levels. Following reltively short period t the MATERIALS AND METHODS therpeuti level, drug onentrtion MATERIALS eventully drops off until re-dministrtion. Tody new methods of drug delivery re possile: desired drug relese n e provided y rte-ontrolling memrnes. The reltively high GI onentrtion nd plsm peks Atorvsttin Clium ws supplied s gift y Pne Bioteh Ltd, Punj, Indi. Eudrgit RS100 nd Eudrgit RL100 ws supplied y Evonik Industries AG Mumi, Indi. Vrious hemils inluding Light Liquid Prffin, n- ssoited with onventionl formultions hexne, etone (Merk Speilties Privte result in n inresed inidene of side effets; Limited, Mumi), nd Methnol ws therefore multiple dily dministrtions were needed 1,2. The purpose of this reserh ws to formulte nd evlute sustined relese mirospheres of torvsttin lium. As the iovilility of otined from Lo Chemils Pvt. Ltd., Mumi, Indi. All other hemil regents were of nlytil grde nd were used without ny further purifition. Distilled wter ws used for ll of the experiments. torvsttin Clium is 13% to 14% owing to mjor hepti first pss metolism. It hs n elimintion hlf life of 13h nd hs n sorption zone from the upper intestinl trt. Effiy of the dministered dose my get diminished due to the inomplete drug relese from the devie ove the sorption zone. Atorvsttin Clium requires twie dy drug METHODS Preprtion of mirospheres A solution of ATC (10 ml ontining 80 mg Drug nd 50 mg sterte) in dihloromethne (oil phse) ws homogenized in eudrgit RS100 nd eudrgit RL100 (240 mg) in 10 ml etone nd methnol mixture (3:2, queous Vol. 2 (2) Apr-Jun

2 phse) using sonitor with pproximtely 2 minutes of sonition t room temperture. The resulting oil-in-wter (o/w) emulsion ws then emulsified t room temperture into (100 ml) of light liquid prffin solution using 0.1% v/v spn 60 emulsifier nd 10% v/v n-hexne s hrdening gent nd stirred with mehnil stirrer t stirring rte of 1000 rpm for 2 h to llow the evportion of the orgni solvent. The hrdened mirospheres were seprted from the oil phse y filtrtion, rinsed with 40 ml of petroleum ether. The wshings were heked for the sene of orgni solvents spetrophotometrilly nd suessive wshings ontinued till this ws hieved, nd vuum dried overnight t room temperture In these studies the effet of the following formultion vrile on the mirospheres size, surfe morphology, drug loding nd enpsultion effiieny, proess yield were investigted: All mirospheres formultions were prepred in triplite. Following re the Polymer:drug rtio: This ws investigted y vrition in the polymer:drug rtio (1:1, 2:1, 3:1, 4:1 nd 5:1, w/w). 1. Nture nd onentrtion of emulsion stilizer in the externl queous phse: While mintining onstnt volume for the externl oil phse, mirospheres were produed using stilizer spn 60 t vrious onentrtion (0.05, 0.1, 0.2% w/v). 2. Volume of n-hexne. 3. Volume of externl oil phse. 4. Stirring rte (500, 1000 nd 1500 rpm). 5. Durtion of gittion during emulsifition (1, 2, 3 h). RESULTS AND DISCUSSION The hydrophoi drugs require the use of o/w/o emulsion solvent evportion method, first the drug is dissolved in oil phse efore dissolving in queous polymer solution. The fst evportion rte of the solvent permits redution in the proessing time; moreover the evportion rte my e used to ontrol the mirospheres size s ompred with other methods where evportion follows the mirospheres formtion. Polymer:drug rtio Atorvsttin Clium loded mirospheres were prepred using using different polymer:drug rtio (from 1:1, 2:1, 3:1, 4:1, 5:1, w/w) y vrition in the weight of polymer dissolved in Aetone nd methnol solution (3:2) to investigte the eventul modifition of the prtile size, drug loding, effiieny of entrpment nd proess yield. Inresing the weight of polymer in fixed volume of orgni solvent resulted in n inrese in men prtile size (from ± 2.14 μm to ± 2.07 μm for 1:1 to 5:1, i.e. inrese of 64.95%). suggesting tht the higher weight of polymer in the smple my hve led to n inresed frequeny of ollision, resulting in fusion of semi-formed prtiles nd produing finlly n overll inrese in the size of the mirospheres.for 1:1 polymer:drug rtio the prtiles otined were spheril in shpe ut with rough surfe nd men dimeter of ± 2.14 μm, n enpsultion effiieny of ± 2.52% (w/w) nd drug loding of ± 1.14 % (w/w). Further inrese in polymer:drug rtio from 2:1 to 4:1 led to the men dimeters of ± 1.92 μm, ± 1.06 μm nd ± 1.12 μm, the enpsultion effiienies of ± 1.33% (w/w), ± 1.67% (w/w) nd ± 2.52% (w/w) nd the drug lodings of ± 1.17% (w/w), ± 1.04% (w/) nd ± 1.06% (w/w), respetively. A further inrese in polymer:drug rtio, i.e., 5:1 led to prodution of spheril prtiles in ggregtes with men dimeters of ± 2.07 μm, n enpsultion effiieny of ± 1.09% (w/w) nd drug loding of ± 2.53% (w/w). Bth Code Tle 1: Effet of polymer:drug rtion on mirospheres hrteristis Polymer: Drug Rtio (w/w) Men Dimeter (µm) ± S.D. Drug Loding (%,w/w) ± S.D. Entrpment Effiieny C (%, w/w) ± S.D. Proess Yield,B AE-1 1: ± ± ± ± 1.38 AE -2 2: ± ± ± ± 2.21 AE -3 3: ± ± ± ± 1.24 AE -4 4: ± ± ± ± 1.98 AE -5 5: ± ± ± ± 1.67 Dt represent the men of three independent experiments. Perentge of weight of miroprtiles reovered with respet to weight of polymer utilized. Perentge of enpsulted drug with respet to the totl mount used Vol. 2 (2) Apr-Jun

3 emulsion solvent evportion method using s dispersion medium light liquid prffin (100 onentrtion(10% v/v) stirring speed: 1000 rpm nd stirring time: 2 h. Conentrtion of n-hexne n-hexne ws seleted s hrdening gent of hoie, it llowed the preprtion of prtiles in the size rnge of ± 1.03 μm with n interesting drug loding of ± 1.14% (w/w). For ll the n-hexne onentrtion in our experimentl onditions, respetive emulsion droplets formed during the gittion seemed to e stle enough to hrden fter solvent evportion nd form the mirospheres. Bth Code Tle 2: Effet of n-hexne onentrtion on mirospheres hrteristis n-hexne Conentrtion (%v/v) Men Dimeter (µm) ± S.D. Drug Loding Entrpment Effiieny C (%, w/w) ± S.D. Proess Yield B H1AE ± ± ± ±1.23 H2AE ± ± ± ±2.05 H3AE ± ± ± ±1.69 Nture nd onentrtion of emulsifier stilizer in the externl oil phse Of the stilizer studied (tween 80, spn 20, spn 60), spn 60 resulted in suessful preprtion of mirospheres. Nevertheless 0.1%w/v spn 60 ws seleted s stilizer of hoie, sine it llowed the preprtion of prtiles in the size rnge of ± 1.53 μm with n interesting drug loding of ±. 2.14% (w/w). Exept when 0.1% spn 60 is used, it ould e shown tht hnges in spn 60 onentrtion were devoided of the effet on drug entrpment, nd prtile size distriution. For ll the spn 60 onentrtion the respetive emulsion droplets formed during the gittion seemed to e stle enough to hrden fter solvent evportion nd form the mirospheres Bth Code Tle 3: Effet of Emulsifier onentrtion on mirospheres hrteristis Emulsifier Con. (%v/v) Men Dimeter (µm) ± S.D. Drug Loding Entrpment Effiieny,C (%, w/w) ± S.D. Proess Yield,B E1AE ± ± ± ±1.83 E2AE ± ± ± ±2.11 E3AE ± ± ± ±1.58 Dt represent the men of three independent experiments. Perentge of weight of miroprtiles reovered with respet to weight of polymer utilized. Perentge of enpsulted drug with respet to the totl mount used emulsion solvent evportion method using s dispersion medium light liquid prffin (100 onentrtion(10% v/v) stirring speed: 1000 rpm nd stirring time: 2 h. Oil:Aqueous phse rtio In phse rtio, different volumes of light liquid prffin (150, 200, 300 ml) were employed s externl dispersing phse, resulting in different rtios etween queous externl nd oil internl phses (o/w rtio), nmely 15:1, 20:1, 30:1. The polymer:drug rtio ws 3:1. The use of the lower w/o rtio (15:1, i.e., 150 ml) led to formtion of irregulr mirospheres with men dimeter of ± 1.64 μm, n enpsultion effiieny of ± 1.12% (w/w) nd drug loding of ± 1.64% (w/w). The highest w/o rtio (30:1, i.e., 300 ml) led to ggregtes of prtiles fter isoltion. Conversely prtiles produed y 20:1 w/o rtio (200 ml) enled the prodution of spheril mirospheres with men dimeter of ± 2.03 μm, the enpsultion effiieny of ± 1.72% (w/w) nd drug loding of ± 1.08% (w/w). Vol. 2 (2) Apr-Jun

4 Bth Code Tle 4: Effet of Oil:queous phse rtio on mirospheres hrteristis Oil:queous rtio Men Dimeter (µm) ± S.D. Drug Loding (%w/w)± S.D. Entrpment Effiieny,C (%,w/w)± S.D. Proess Yield,B (%w/w)± S.D. P1AE-3 15: ± ± ± ±2.05 P2AE -3 20: ± ± ± ±1.97 P3AE -3 30: ± ± ± ±1.68 Dt represent the men of three independent experiments. Perentge of weight of miroprtiles reovered with respet to weight of polymer utilized. Perentge of enpsulted drug with respet to the totl mount used emulsion solvent evportion method using s dispersion medium light liquid prffin (100 onentrtion(10% v/v) stirring speed: 1000 rpm nd stirring time: 2 h. Stirring speed Stirring speed plys n importnt role in the mirospheres size distriution nd drug loding. In ft using 3:1 polymer:drug rtio, 20:1 o/w rtio it ws found tht 500 rpm stirring speed produed prtiles with rough nd irregulr surfe. On the ontrry, triple stirring speed, nmely 1500 rpm, led to the prodution of spheril mirospheres, hrterized y ± 2.12 μm men dimeter, ±1.18% (w/w), drug loding ± 2.18% (w/w) nd ± 1.43% (w/w) enpsultion effiieny. The est results in term of proess yield were otined y the use of 1000 rpm stirring speed (96.85 ± 1.43%, w/w), mirospheres in this ondition were spheril, with ± 1.83 μm men dimeter, drug loding ± 1.92% (w/w) nd ± 1.09% (w/w) enpsultion effiieny. Bth Code Tle 5: Effet of stirring speed on mirospheres hrteristis Stirring Speed (rpm Men Dimeter (µm) ± S.D. Drug Loding (%W/W) ± S.D. Entrpmen t Effiieny, C (%, W/W) ± S.D. Proess Yield,B (%W/W) ± S.D. T1AE ± ± ± ±1.02 T2AE ± ± ± ±1.18 T3AE ± ± ± ±1.74 Dt represent the men of three independent experiments. Perentge of weight of miroprtiles reovered with respet to weight of polymer utilized. Perentge of enpsulted drug with respet to the totl mount used emulsion solvent evportion method using s dispersion medium light liquid prffin (100 onentrtion(10% v/v) stirring speed: 1000 rpm nd stirring time: 2 h. Stirring Time For onstnt speed of 1000 rpm, polymer:drug rtio of 3:1, o/w rtio of 20:1 n inrese of the stirring time from 1 to 3 h Bth Code resulted in redution in mirospheres size (from ± 2.13 to ± 2.72 μm). These oservtions ould e explined y the inresed sher stress generted in the emulsions ssoited to the inrese in the durtion of gittion t high homogeniztion rtes tending to divide the droplets of the emulsions nd finlly induing derese in the men prtile size. A 2 h stirring time ws hosen euse the entrpment effiieny ws higher (73.48 ± 1.09%, w/w) thn fter 2 h (67.18 ± 1.43%, w/w). Tle 6: Effet of stirring time on mirospheres hrterstis Stirring Time (hrs) Men Dimeter (µm) ± S.D. Drug Loding (%W/W) ± S.D. Entrpment Effiieny,C (%, W/W) ± S.D. Proess Yield,B (%W/W) ± S.D. T1AT ± ± ± ±1.09 T2AT ± ± ± ±1.29 T3AT ± ± ± ±1.82 Dt represent the men of three independent experiments. Perentge of weight of miroprtiles reovered with respet to weight of polymer utilized. Perentge of enpsulted drug with respet to the totl mount used Vol. 2 (2) Apr-Jun

5 emulsion solvent evportion method using s dispersion medium light liquid prffin (100 onentrtion(10% v/v) stirring speed: 1000 rpm nd stirring time: 2 h. Prtile size nlysis The prtile size nd size distriution of the prepred mirospheres were mesured y lser diffrtion in prtile size nlyzer (Mstersizer, Mlvern Instruments, UK). The dried powder smples were suspended in deionised wter nd sonited for 1 min with n ultr-sound proe efore mesurement. The otined homogeneous suspension ws determined for the equivlent volume dimeter nd triplite mesurements were mde for eh th of mirospheres. FT-IR Spetrosopy studies Infrred (IR) spetrosopy ws onduted using FTIR, ALPHA, Bruker nd the spetrum ws reorded in the wvelength region of 4000 to 500 m 1. FTIR nlysis reveled tht there ws no intertion etween the drug nd the polymer, thus these polymers n e onveniently used in further development of sustined relese torvsttin lium mirospheres. Further FTIR studies indited tht the drug rystllinity did not hnge upon frition. There ws no formtion of morphous drug upon frition. Bnds due to ronyl ond strethes were studied to infer the physil stte of the drug. FTIR dt in the region ( m -1 ) were ompred for pure torvsttin lium nd torvsttin lium enpsulted in the mirospheres. As expeted, two min hrteristi C=O nds were oserved for rystlline drug t 1653 nd 1590 m 1, ssigned to the liphti nd romti ronyl strething nds, respetively. Mirosphere formultion lso yielded peks t the sme wvelengths. The FT-IR spetr of pure Atorvsttin Clium nd mirospheres of the optimized th (FOAT-3) re shown in Figures 1 () & 1 (). The FT-IR spetr of pure ATC showed hrteristi peks t m -1 (C-H - strething), m -1 (C-N - strething), m -1 (C-HO - strething loholi group), m -1 (C=O - strething midi group), m -1 (N-H - strething), m-1 (C=C - ending), m -1, m -1 (C-F - strething), m -1 (O-H - ending). It might e the possiility of intermoleulr hydrogen onding etween djunt ATC moleules. The spetrum of pure drug ws equivlent to the spetr otined y the optimized th (FOAT-3). This indited tht no intertion ourred with miroenpsultion of drug nd polymers. The results reveled no onsiderle hnges in the IR peks of drugthese oservtions indited the omptiility of polymers nd the drug. Fig. 1(): FTIR Grph of Drug (Atorvsttin Clium) Figure 1() FTIR Grph of Optimized Bth FOAT-3. Vol. 2 (2) Apr-Jun

6 Powder X-ry diffrtion (pxrd) for ssessment of rystllinity In order to determine the physil stte of the drug efore nd fter mirospheres preprtions, the XRD ptterns of the pure drug nd the formultions were investigted using n Bruker s D8 dvne diffrtometer (Germny). The smples were irrdited with monohromtized CuKα rdition, nd the snning rnge (2θ) ws from The voltge nd urrent were set to 30kv nd 30mA, respetively. X-ry ptterns were nlyzed using n X-pert dt olletor nd X- pert dt viewer V-1.0 softwre. The diffrtion pttern of pure drug showed hrteristi high-intensity diffrtion peks t 8.96, 9.35, 10.01, 10.37, 11.65, 12.00, 16.85, 19.26, 21.36, 22.50, 23.12, nd 23.51, whih indites tht the drug is present in the rystlline form tht is lso onfirmed y DSC results, wheres FOAT-3 mirospheres formultion showed refletions t 17.18, 18.66, 21.08, nd The pure drug exhiits refletions t 8.9, 9.3 nd 10, these strong refletions of pure drug were msked in the mirospheres formultion nd exhiits wek refletions t 8.9, 9.3 nd 10 (two thet) 2θ. This derese in intensity of refletion is ttriuted to the presene of other exipients in the formultion, Figure 2 (). It is ler tht the refletions of the pure drug (Figure 2 () mth stisftorily with the refletions of the drug in the mirosphere formultion. Thus, it n e onluded tht the polymorph of pure drug ws the sme s tht of Atorvsttin Clium polymorph inorported in mirospheres, nd no trnsformtion took ple during the mnufturing proess nd storge. Fig. 2(): XRD Pttern of Pure Drug (Atorvsttin Clium) Figure 2() XRD Pttern of Optimized Bth FOAT-3 In vitro relese studies In vitro dissolution studies were rried out on the mirospheres t 37 o C (± 0.5 o C) t 100 rpm with USP Dissolution Apprtus II (). For the id stge, n urtely weighed smple of miroprtiles ws suspended in the dissolution medi onsisting of 500 ml of 0.1 N (ph 1.2) hydrohlori id without enzymes nd dissolution ws done for 2 h. At the end of the 2 h, 400 ml of 0.1M trisi sodium phosphte ws dded to ll dissolution vessels, the ph ws djusted to 7.2 (± 0.2) nd the dissolution ws ontinued until the mirospheres were depleted of drug or for 24 h. Aliquots of dissolution fluid were withdrwn t speified time intervls to ssy the relesed drug spetrophotometrilly t 246 nm in oth stges of dissolution. Eh grphil dt point ws n verge of dissolution dt from three smples. Corretions were mde for the removl of smples (Figure 3). Vol. 2 (2) Apr-Jun

7 Cumultive Perentge Drug Relese of Vrious Bthes 100 Cumultive % Drug Relese AT-1 AT-2 FOAT-3 AT-4 AT-5 Time (h) Fig. 3: Relese profile of Atorvsttin Clium from Eudrgit RS 100 nd Eudrgit RL 100 mirospheres Drug relese pttern from mirospheres In order to investigte the relese mehnism of present drug delivery system, the relese dt of miroprtiles were fitted to lssi drug-relese kinetis models. The relese rtes were nlysed y zero-order model, first order model, Higuhi model nd Korsmeyer Pepps model, whih hve een suggested to desrie drug-relese kinetis from miroprtiles. Higuhi plot nd Pepps plot of finl optimized th of mirospheres (FOAT- 3) re given in Figure 4() nd 4() respetively. % C u m u l t i v e D r u g R e l e s e Higuhi Plot y = x R 2 = FOAT-3 40 Liner (FOAT-3) Squre Root of Time (hrs) Log ulmultive relese (%) Korsmeyer Pepps plot Log of time (h) y=0.587x R 2 = log of FOAT-3 Liner (logof FOAT-3) Fig. 4(): Higuhi Plot Fig. 4(): Korsemeyer Pepps Plot Vol. 2 (2) Apr-Jun

8 Kinetis of drug relese In order to investigte the relese mehnism of present drug delivery system, the dt otined from in vitro relese of finl optimized th were fitted into equtions for the zeroorder, first-order, nd Higuhi relese model nd Pepps eqution. The interprettion of dt ws sed on the vlues of the resulting regression oeffiients. The in vitro drug relese showed the regression oeffiient vlues for Higuhi s model (Figure 4) (R 2 = ) nd Pepps model (R 2 = ) nd vlue of n = (Figure 4) inditing nomlous trnsport. Sttistil nlysis Sttistil dt nlysis were performed using the Student s t-test nd one-wy nlysis of vrine (ANOVA) with p < 0.05 s the miniml level of signifine. Snning eletron mirosopi nlysis The prepred miroprtiles were oted with gold plldium under n ir tmosphere for 150 seonds to hieve 20 nm film (Coter Polron,18mA urrent t 1.4 kv).the oted smple were then exmined using snning eletron mirosope(vrile Pressure Snning Eletron Mirosope (SEM) Hithi S3400N). The morphology nd pperne of miroprtiles were exmined y snning eletron mirosopy s shown in figure 5. Fig. 5: SEM Imges of Mirospheres of the optimized th CONCLUSION O/W/O emulsion solvent evportion method ws used to prepre mirospheres of Atorvsttin Clium with Eudrgit RS100 nd Eudrgit RL100. These investigtions hve lso provided n understnding of the effets of some proess prmeters on prtile size nd shpe, nd enpsultion effiieny, drug loding nd proess yield The investigted system hs the potentil to remin in treted site for prolonged periods nd ple of mintining onstnt onentrtion of drug through longer durtion of time due to its sustined tion, This n e expeted to redue the prolem of low iovilility nd high first pss metolism nd lso derese the dose dependent side effets ssoited with repeted dministrtion of onventionl Atorvsttin Clium loded dosge forms, whih ultimtely improve ptient ompline. O/W/O emulsion solvent evportion method were suitle for the preprtion of mirospheres in the size rnge of ±1.06 μm, the enpsultion effiieny ws ± 1.21% (w/w), drug loding ws ± 1.04% (w/w) nd the proess yield ws ± 1.24% (w/w). It onluded tht sustined relese Eudrgit RS100 nd Eudrgit RL100 mirosphere ontining Atorvsttin Clium were suessfully prepred y using w/o/w emulsion solvent evportion method with the seletion of pproprite experimentl onditions. REFERENCES 1. Bjj A, Desi M; Chllenges nd strtegies in novel drug delivery tehnologies. Phrm Times. 2006;38(4): Rvi Kumr M.N.V.; Nno nd miroprtiles s ontrolled drug delivery devies. J. Phrm. Phrmeut.Si. 2000;3(2): Kdu PJ, Kushre SS, Thker DD, Gttni SG. Enhnement of orl iovilility of torvsttin lium y self-emulsifying drug delivery systems (SEDDS). Phrm Dev Tehnol. 2011; 16: Dimitr S, Christos GK. Identifition nd quntittive determintion of Atorvsttin lium polymorph in Vol. 2 (2) Apr-Jun

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