Prep aration,characterization,and in vitro/vivo evaluation of p olym er-assisting form ulation of atorvastatin calcium based on solid d ispersion technique

Wenxiang Dong,Xitong Su,Meng Xu,Mingming Hu,Yinghua Sun,Peng Zhang

Keywords:Atorvastatin calcium Poloxam er 188 Solid dispersion Dissolution rate Oral bioavailability

A B S T R A C T Due to low solubility and bioavailability,atorvastatin calcium is confronted w ith challenge in conceiving appropriate formulation.Solid dispersion of atorvastatin calcium w as prepared through the solvent evaporation m ethod,w ith Poloxam er 188 as hydrophilic carriers.This form ulation w as then characterized by scanning electron m icroscopy,differential scanning calorim etry,powder X-ray diffraction and fourier transform infrared spectroscopy.Moreover,all these studies suggested the conversion of crystalline atorvastatin calcium.In addition,the drug solubility studies as well as dissolution rates com pared w ith bulk drug and m arket tablets Lipitor w ere also exam ined.Furtherm ore,the study investigated the pharm acokinetics after oral administration of Lipitor and solid dispersion.And the AUC0–8 h and Cmax increased after taking ATC-P188 solid dispersion orally com pared w ith that of Lipitor.All these could be dem onstrated that ATC-P188 solid dispersions w ould be prospective m eans for enhancing higher oral bioavailability of ATC.

1. Introd uction

Atorvastatin calcium(ATC)is indicated in the treatm ent of atherosclerosis and coronary disease alone or along w ith other lipid-low ering medicine[1].It reduces plasma cholesterol levels since it inhibited the synthesis of HMG-Co A reductase and cholesterol[1,2].ATC is also helpful in increasing the receptor of low density lipoprotein receptor on cell surface and decrease triglyceride levels in serum,meanw hile it can increase the level of high density lipoprotein(HDL)[1,3].Ow ing to its low solubility and f irst-pass m etabolism,the oral bioavailability is only around 14%[4,5].Therefore,development of ATC formulation in virtue of low solubility and oral bioavailability is challenging[6].Am ong those technologies includingtechnique[8–12],salt form ation [13,14],nanosuspension[15]and other techniques,solid dispersion technology has received popularity as it increases solubility of insoluble drugs[16–18].The m ost im portant feature of solid dispersion technology is that drug w as highly dispersed in suitable carriers[19].The techniques include tw in screw extrusion,melting m ethod,spray-dried dispersion,solvent evaporation m ethod and other m ethods[20].Solid dispersion could enlarge the surface of the drug particles,w hich results in enhancing the drug release based on Noyes–Whitney equation[10,21].Moreover,the existence of P188 not only ensures the high dispersion of the drug,but also could effectively prevent the aggregation of the atorvastatin calcium.On the other hand,oral bioavailability of crystalline ATCcould be enhanced by converting the crystalline state and particle property of drug[6].

Poloxam er 188(P188)is a kind of non-ionic surfactant approved by FDA,com m only used w ith insoluble drugs as solubilizer and surfactant,based on high drug loading,low m elting point,hydrophilicity and safety.For exam ple,it has been used in therm oreversible gels for topical drug delivery as compatibility w ith skin w hich could increase skin perm eability and prom ote the absorption of external agents.It has been reported that P188 can play eff icient role on anti-tum or m echanism w hen used as carriers for drug delivery[22,23].

This study is mainly aimed at enhancing the solubility,dissolution rates and oral bioavailability of ATC by conventional solvent evaporation m ethod w ith P188 as the carrier.Quite a few reports have form ulated SDs of ATC using PEG 6000/4000,PVPK30,Soluplus[21,24–26]and so on as carriers.How ever,they have lim itations com pared w ith P188 due to the high viscosity,w hich lead the solution diff icult to be desiccated.P188 is w idely used in various form ulations as pharmaceutical excipient based on promoting drug absorption and non-toxic effects.The lim itation of the above m entioned m ethods(hot-m elt m ethod,spray-dried m ethod)is that these m ethods require extra instrum ents and a large am ount of drugs or carriers.The spray-dried method includes interaction of the m achine conf iguration and formulation variables,w hich could affect drying eff iciency and therefore im pact the solid states property of SD.How ever,the hot-m elt m ethod com m only operates at high tem peratures(m ore than 100°C),w hich could affect the stability of drugs.By com parison,the conventional solvent evaporation m ethod has the advantages of low cost,operating and reproducing conveniently.In addition,it is reported that P188 and PEG 4000 can increase the release of the ATC w hen they as the carriers of solid dispersion sim ultaneously and it has not found solid dispersion of ATC using P188 as carriers alone updated.Compared w ith the tw o carriers,the prescription in this w ork is sim pler and the process is easier to reproduce.In this w ork,the physiochem ical characterization of SD w ere detected by scanning electron microscopy(SEM),differential scanning calorimetry(DSC),pow der X-ray diffraction(PXRD),and Fourier transform infrared spectroscopy(FTIR)after preparation.Som e studies have show n im provem ent of solubility and drug release of ATC through solid dispersion method relative to bulk drug(API)and physical m ixture.Finally,a pharm acokinetic study w as conducted in rats by oral adm inistration.In the existing literature,m any form ulations including solid dispersion prepared by atorvastatin calcium has only increased bioavailability relative to the API.Only one report is found update,pharm acokinetics(PK)results revealed that the ATC–Nanoparticles(ATC–NPs)form ulations w ere of signif icantly low er bioavailability com pared to Lipitor although pharm acodynam ics(PD)results revealed that Lipitor and ATC–NPs formulation w ere equally effective in reducing levels of low density lipoproteins and triglycerides[27].In order to validate the form ulation,the dissolution and bioavailability of solid dispersion w as compared w ith Lipitor(10 m g)in this w ork.

2. Materials and m ethods

2.1. Materials

Atorvastatin calcium bulk drug was purchased from Zhejiang New Donggang Pharm aceutical Co.,Ltd.(China).P188 w as given by BASF Co.,Ltd.(Shanghai,China).The com m ercial product(Lipitor,10 m g dose)w as purchased from Pf izer Co.,Ltd(Dalian,China).Methanol w as bought from Tianjin Concord Technology Co.,Ltd.(China).

2.2. Preparation of the solid dispersion

Different atorvastatin calcium bulk drug:polym er(P188)combinations(1:1,1:3,1:5,1:8;w/w)w ere dissolved in m ethanol(25 m l),ultrasonic for 5 m in and stirred for 30 m in at 40°C by using water bath.The rotary evaporator w as used to evaporate m ethanol at 35°Cafter com plete dissolving.Then it w as transferred to a vacuum drying apparatus to rem ove residual solvent for 24 h.The resultant w as pulverized,f iltrated through 80 mesh sieve and stored in a desiccator at about 25°C.

2.3. Preparation of physical mixture

Different w eight ratios of drug:P188(1:1,1:3,1:5,1:8;w/w)w ere prepared in m ortar until sym m etrical m ixture w ere form ed.The resulting m ixture w as also f iltrated through 80 m esh sieve and stored in a desiccator at about 25°C.

2.4. Optimization of solid dispersion

In order to evaluate the feasibility of solid dispersion technique,it is unavoidable to investigate the dissolution behavior[28].Hence,the appropriate dissolution m edium is critical.The solubility of atorvastatin calcium increased w ith the enhancem ent of p H and p H 1.0 buffer was used as a tool for selecting form ulation of solid dispersion according to relevant literature[29].Ultraviolet-visible spectroscopic m ethod w as developm ent to analyze the dissolution study.It w as carried out w ith dissolution apparatus(RC806D dissolution apparatus,Tianda Tianfa Technology Co.Ltd.,Tianjin,China)using the paddle m ethod.Solid dispersion(equivalent to 10 m g of atorvastatin)w as placed into 900 m l of dissolution m edium(p H 1.0)at 37±5°C and paddle rotation speed w as 50 rpm.10 m l of solution w as sam pled at a predeterm ined interval(5,10,20,30,45,60 m in)and an equivalent volum e(10 m l)of prewarm ed fresh media(37°C)was added in each vessel to keep equivalent of volum e.The concentration of ATCw as analyzed by an ultraviolet spectrophotom eter(UV1102 II spectrophotom eter,Tianm ei Technology Co.Ltd.;Shanghai,China)at a wavelength of 244 nm.All sam ples were performed in triplicate and no adsorption of ATC to the f ilter m em branes could be detected.

2.5. Solubility of solid dispersion

Excessive of the physical m ixture(PM),and the SD pow der w as added in test tube containing 10 m l dissolution m edium:0.1 M HCl,acetate buffer solution(p H 4.5),phosphate buffer solution(p H 6.8 and 7.2)and w ater.These sam ples w ere placed in w ater bath at 37±0.5°C for 48 h w ith vortex m ixing.The suspensions w ere centrifuged at 13,000 rpm for 5 m in and f iltrated through a 0.45μm membrane f ilter.After dilution,the sam ples w ere analyzed at a w avelength of 244 nm by an ultraviolet spectrophotom eter[21].No effect of polym er on UV m easurem ent could be detected.

2.6. Characterizations of solid dispersion

2.6.1. Fourier-transform infrared spectroscopy

FTIR spectrum of sample w as obtained on IFS-55 system(Bruker Corporation,Sw itzerland)using KBr disc m ethod.The sam ple w as recorded over w ave num ber range of 4000–400 cm-1at the resolution of 2.0 cm-1.

2.6.2. Powder x-ray diffraction

The pow der X-ray diffraction patterns of the sam ples w ere recorded using Rigaku Minif lex diffractm eter(Rigaku Corporation,Tokyo,Japan),a voltage of 40 k V and a 30 m A current.The sam ple w ere analyzed over a 2θrange of 5–45°,w ith a scanning rate of 2°/m in and a Cu Kαradiation source.

2.6.3. Differential scanning calorimetry

Differential scanning calorim etry m easurem ent w as conducted by DSC-1(Mettler-Toledo International Inc.,Sw itzerland)w ith cooling equipm ent.The tem perature program w as performed form 30 to 250°Cat a heating rate of 10°C/m in.

2.6.4. Scanning electron microscopy

Scanning electron m icroscopy(S-3400,Hitachi,Tokyo,Japan)w as applied to observe the m orphology of bulk drug,P188,physical m ixture and solid dispersion.The samples were f ixed using m utual conductive adhesive tape on alum inum stubs and sputter-coated w ith a gold layer at 20 m A for 30 s in an ion sputter coater(S-570,Hitachi,Tokyo,Japan)at a pressure of 8–10 Pa prior to the observation at an accelerating voltage of 20 k V.

2.7. Pharmacokinetics studies

Ten Wistar rats(m ale,220–250 g),w hich w ere provided by the Anim al Center in Shenyang Pharm aceutical University,w ere kept under standard laboratory conditions at a tem perature of 25±2°Cand relative hum idity(55%±5%).The rats w ere divided into tw o groups:A(Lipitor,10 m g)and B(solid dispersion)at a dose of 25 m g/kg,random ly.

The Lipitor and solid dispersion were grinded into powder w hich dispersed in 0.4%Carboxym ethylcellulose sodium(CMC–Na)before adm inistration.Blood sam ples(0.3 m l)w ere collected w ithin 8 h from orbital plexus and added into heparinized tubes at 0.083,0.167,0.333,0.5,0.75,1,1.5,2,4,6 and 8 h after oral adm inistration.The sam ples w ere centrifuged at 13 000 rpm for 10 m in instantly and the separated plasm a w as stored at-20°C for analysis.

2.8. Plasma sample analysis

First,50μl acetonitrile and 50μl Gliclazide solution(4μg/m l)w ere added into 100μl plasm a sam ple and vortex-m ixed for 3 m in.After that,2 m l acetonitrile w as added to vortexed for about 5 m in and centrifuged at 3500 rpm for 10 m in.The supernatant w as evaporated to dryness at 37°Cunder nitrogen.The residue w as reconstituted w ith 100μl of acetonitrile.The samples(20μl)w ere chrom atographed on a reverse phase Phenom enex Ultrem ex C18(250 m m×4.6 m m,5μm)at a w avelength of 244 nm.Chrom atographic analysis w as carried out on a HPLC system(HITACHI,Japan),consisting of a quaternary pum p,an autosam pler,detector and a colum n oven.The m obile phase consisting of acetonitrile and 0.5%form ic acidw ater solution in the ratio of 48:52(v/v)w as f iltered through a 0.22μm membrane f ilter and degassed under vacuum before use.The concentration of each sam ple w as calculated referred to a calibration curve w ith the concentration range from 0.075 to 2μg/m l.The related pharm acokinetic param eters w ere analyzed using DAS 2.0 softw are[30].

3. Result and d iscussion

3.1. Optimization of solid dispersion

Dissolution behavior is a signif icant m ean to guide the developm ent of new formulation and could be used as a distinguishing m ethod in form ulation selection[31,32].According to related literature,the dissolution w as carried out in p H 1.0 m edia solution.The results w ere calculated from standard calibration curve(A=0.048C–0.042,R2=0.999,the range of 2–20μg/ml)of ATCand the cumulative percentage release of the drug w as plotted against tim e.Based on the dissolution prof ile(Fig.1),the release of ATC increase subsequently as the proportion of P188 increased(1:1,1:3 and 1:5).How ever,there was no signif icant difference between the releases of solid dispersion w ith drug:polym er ratio of 1:8 and the ratio of 1:5.In view of solvent quantity and the ratio of carrier,solid dispersion w ith drug:polym er ratio of 1:5 w as selected for further study.To evaluate this formulation,dissolution test was also carried out at different dissolution m edium(p H 1.0,p H 4.5,p H 6.8 and w ater)to com pare dissolution prof ile betw een bulk drug and Lipitor.As depicted in Fig.2,The release of bulk drug was 40%in p H 1.0 media over 60 min.Nevertheless,either m arket tablets or the solid dispersion exhibited a signif icant enhancem ent in drug release than that of bulk drug.In other m edia,about 100%ATC w ere released from solid dispersion and Lipitor,higher than bulk drug w ithin 30 m in.It is w orthy of note that the release rates w as quite slow er in Lipitor compared to solid dispersion,w hich could be seen w ithin 5 m in.

Fig.1–Dissolution p rof iles of d ifferent w eight ratios(1:1,1:3,1:5,1:8)of drug:P188.Each value rep resents the m ean±SD(n=3).

The enhancement of dissolution rates could be due to m olecularly dispersion of ATC w ithin P188 and drug re-crystallization during preparation[21,28].Sim ilar results have been reported for celecoxib[29],diazepam[33],and felodipine[34].Hence,the preparation of solid dispersion essentially enhanced the dissolution rate of ATC taking advantage of the increased surface area,am orphous state and effective wettability of P188.

3.2. Solubility of SD

Based on the results(Fig.3),the im proved solubility of ATCin physical m ixture m ight be result from the hydrophilic nature of P188.How ever,the results of further increase solubility in SD revealed that the solid dispersion techniques caused further increase in ATCsolubility compared to the bulk drug and PMs.

3.3. Characterizations of SD

3.3.1. Fourier-transform infrared spectroscopy

Fig.2–Dissolution p rof iles of d ifferent m ed ium p H 1.0(A),p H 4.5(B),w ater(C)and p H 6.8(D)of bulk drug,solid disp ersion and Lipitor.Each value rep resents the mean±SD(n=3).

If the drugs have different crystal form,there m ay be difference in chem ical bond length and angle,w hich could affect vibrational-rotational transitions and some characteristics such as IR absorption band frequency,peak shape,peak position and intensity.Rem arkably,IR spectrum could provide this information about chemical bonds,characteristic function groups and generally detect possible m olecular interaction betw een drugs and carriers in the solid dispersion system[21].In this study,FITRw as applied to determ ine possible interactions betw een P188 and ATCthrough solvent evaporation m ethod.FT-IR spectrum of ATC,P188,PM,SD are depicted in Fig.4.The bulk drug exhibits characteristic peaks at 3670 cm-1(free O–H stretching vibration),3364.8 cm-1(N–H stretching),3056.0 cm-1(symm etric O–H stretching),2970.4 cm-1(C–H stretching),1650.6 cm-1(asym m etric C=O stretching),1579.1 cm-1(sym m etric C=O stretching),1316.1 cm-1(CH3/CH2deform ation),1241.4 cm-1(C–N stretching),1217.3 cm-1(C-F stretch).The spectrum of P188 show s im portant function groups at 2889.6 cm-1(C–H stretching),1110.9 cm-1(C–O groups).As can be seen,the characteristic of free O–H stretching vibration at 3670 cm-1was absent in SD but appeared in PM,w hich m ight be due to the form ation of am orphous nature of ATC[5].On the basis of FTIR spectrum,som e characteristic function groups of 3364.8 cm-1,3056.0 cm-1in the spectrum of SD disappeared but there are characteristic peaks at 3364.7 cm-1,1650.6 cm-1,1579.9 cm-1in the spectrum of physical m ixture,w hich m ight be due to interaction betw een ATCand P188.Meanw hile,in view of chemical structure of P188 and ATC,it could form hydrogen bond w hich can effectively prevent re-crystallization of am orphous drugs and increase stability[21].No additional new peaks is form ed in SD suggested that there w as no chem ical interaction occurring during the preparation process[35].

Fig.3–Solubility of bulk drug,physical m ix ture-bulk drug:P188=1:5 and solid dispersion in d issolution m ed ium.

Fig.4–FTIRspectrum of p hysical m ix ture-bulk drug:P188=1:5,solid d ispersion,P188,ATC.

Fig.5–Pow d er X-ray d iffraction p atterns of physical mixture-bulk drug:P188=1:5,solid dispersion,P188,ATC.

Fig.6–Differential scanning calorimetry of physical m ix ture-bulk drug:P188=1:5(A),solid d ispersion,P188,ATC.

Fig.7–SEM im ages of P188(A),solid disp ersion(B),ATC(C)and p hysical m ix ture-bulk drug:P188=1:5(D).

3.3.2. Powder X-ray diffraction

Pow der X-ray diffraction could provide further verif ication of drug crystal conversion.The PXRD patterns of bulk drug,P188,physical m ixture and solid dispersion w ere depicted in Fig.5.The untreated bulk drug exhibited sharp and intense diffraction peaks at 2θvalues of 9.1,9.4,10.2,10.5,11.8,12.1,17.0,19.4,21.5,21.9,22.6,23.3,23.7,24.3,28.8 and 29.1,w hich are consistent w ith the characteristic diffraction peaks of the crystalline Form Idescribed in the patent[36].The bulk P188 shows intense characteristic peaks at diffraction angle(2θ)of 19.1,23.3.Physical m ixtures show ed peaks coincident w ith P188,and som e diffraction peaks at 2θvalues of 9.1,9.4,10.2,10.5,11.8,12.1,17.0,21.6,29.0 are sim ilar to bulk drug although at low intensity(considering the P188 dilution effect,the drug peaks alm ost disappears due to the high percentage of P188).However,no characteristic peaks corresponding to bulk ATC w ere found in solid dispersion.These results indicated that the drug have been transform ed from crystalline Form I to am orphous states.

3.3.3. Differential scanning calorimetry

Fig.6 show s differential scanning calorimetry of bulk drug,P188,physical m ixture and solid dispersion.The DSC curve of P188 displayed a sharp endotherm ic peak at 53°C due to its m elting point.DSC curve of ATC show s tw o endotherm ic peaks,a broad peak at 80–120°C is related to w ater loss or dehydration(dem onstrates the presence of tri-hydrate bulk drug),follow ed by another broad peak at 160°C corresponding to the m elting point of the atorvastatin calcium.Meanw hile,the third broad peak at 210–250°Cmay be attributed to degradation product of ATC[37].Physical m ixture of ATC:P188 w ith the ratio of 1:5 show endotherm ic peaks at 53°C,80–120°C,160°Corigin from the m elting point of P188,w ater loss or dehydration and the m elting point of ATC respectively.No endotherm peaks related to the bulk drug appeared in the SD suggested the conversion of drug crystallinity.According to Fourier-transform infrared spectroscopy,PXRD studies,the ATC has transform ed to am orphous state.

Table 1–Inter-d ay,intra-day p recision and accuracy results for QC sam ples of atorvastatin in rats p lasm a(n=6).

Table 2–Pharm acokinetic p aram eters of ATC in rats after oral adm inistration of Lipitor and solid disp ersion at a d ose of 25 m g/kg(m ean±SD,n=5).

3.3.4. Scanning electron microscopy

The surface m orphology of bulk drug,P188,physical m ixture and solid dispersion w as exam ined by SEM analysis,as depicted in Fig.7.The bulk drug dem onstrated needle-like crystallites w ith sm ooth surface and various particle sizes.Carriers existed as various size of spherical w ith sm ooth surfaces.In physical m ixture,ATC crystals w ere clearly show ed together w ith P188 particles w hich were the same as bulk drug m icrograph.Meanw hile,the m icroscopy of carrier w as converted to irregularly shape particles result from shattering and sieving.On the other hand,no visible needle-like crystallites were w itnessed such as bulk drug micrograph in solid dispersion.In m icrograph of solid dispersion,w e can observe the irregularly shaped particles of carrier.And the sm ooth surface of carrier w as covered by m any sm all lam ellas and particles result from bulk drug.All these suggested that ATC has converted to am orphous form through solid dispersion technique.

3.4. In vivo pharmacokinetics

Ten rats adm inistrated w ith Lipitor and solid dispersion in a dose of 25 m g/kg,respectively.The methodology w as conform ed and the tested linearity w as ranging from 0.075 to 2μg/m l w ith correlation coeff icient of 0.9971 and the standard curve w as Y=2.886X+0.1515.The results for the interday,intra-day precision and accuracy are depicted in Table 1.The m ean extraction recoveries for atorvastatin at 0.2,0.6,1.6μg/m l w ere 94.08%,85.31%,96.94%,respectively.In summ ary,the m ethod is suitable for the determ ination of atorvastatin in plasma.

Fig.8–Mean p lasm a concentration-tim e curves of ATC in rats after oral ad m inistration of Lip itor and solid d ispersion at a d ose of 25 m g/kg AC(m ean±SD,n=5).

The pharm acokinetics param eters are show n in Table 2 and Fig.8.It w as found that the AUC0–8hof solid dispersion(919.0 ng/h/m l)represents greater im provem ent than that of Lipitor(534.5 ng/h/m l).In addition,It w as also observed that AUC0–8hof solid dispersion w as alm ost 1.71-fold com pared w ith Lipitor.It w as obtained that Cmaxof solid dispersion formulation w as 972.2 ng/ml and thus the difference was highly signif icant(P＜0.05)com pared w ith Cmaxof Lipitor.The Tmaxof solid dispersion-the tim e that the plasm a concentration of atorvastatin reaches the m axim um after adm inistrating w ith solid dispersion and Lipitor w as faster than that of Lipitor.The hydrophilicity of ATC increases through solid dispersion technique,w hich is easier to diffuse to hepatocytes.The m etabolism of atorvastatin occurs principally in the liver via cytochrom e P450(CYP)system.All these could explain that the T1/2of solid dispersion formulation was shorter than that of Lipitor[38].As the plasm a concentration and AUC0–8hincreasing w hen adm inistered solid dispersion,w hich indicated that the pharm acological activities of ATC m ay be increased[39].In short,these results suggested that solid dispersion could be applied as an effective form ulation for enhancing the oral bioavailability of ATC[20].

4. Conclusion

The present study dem onstrated the preparation of ATCP188 solid dispersion by conventional m ethod successfully.After com parison of dissolution prof ile betw een various carriers,P188 w as selected as the f inal hydrophilic carrier.The physiochem ical characterization indicated that the drug has dispersed in carriers and transform ed to am orphous state.Solubility and dissolution rates were enhanced signif icantly com pared w ith bulk drug.Meanw hile,the dissolution prof ile of solid dispersion reached the degree of m arket tablets Lipitor.The pharmacokinetic study indicated that the Cmaxand AUC0–8hof solid dispersion w ere im proved nearly 2.87-fold and 1.71-fold com pared w ith Lipitor,separately.It’s therefore reasonable to point out that ATC-P188 solid dispersion using solvent evaporation method could be an effective method for increasing the oral bioavailability of ATC.

Declaration of interest

The authors report no conf licts of interest.The authors alone are responsible for the content and w riting of this article.

Supp lem entary m aterials

Supplem entary m aterial associated w ith this article can be found,in the online version,at doi:10.1016/j.ajps.2018.08.010.