physicochemical characterisation, drug polymer dissolution and in vitro evaluation of phenacetin and...

Physicochemical Characterisation, Drug Polymer Dissolutionand in Vitro Evaluation of Phenacetin and Phenylbutazone SolidDispersions with Polyethylene Glycol 8000

SHERAZ KHAN,1 HANNAH BATCHELOR,2 PETER HANSON,1 YVONNE PERRIE,1 AFZAL R. MOHAMMED1

1Aston Pharmacy School, Aston University, Birmingham B4 7ET, UK

2Heart of England NHS Foundation Trust, Birmingham B9 5SS, UK

Received 11 March 2011; revised 12 April 2011; accepted 20 April 2011

Published online 10 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22613

ABSTRACT: Poor water solubility leads to low dissolution rate and consequently, it can limitbioavailability. Solid dispersions, where the drug is dispersed into an inert, hydrophilic polymermatrix can enhance drug dissolution. Solid dispersions were prepared using phenacetin andphenylbutazone as model drugs with polyethylene glycol (PEG) 8000 (carrier), by melt fusionmethod. Phenacetin and phenylbutazone displayed an increase in the dissolution rate whenformulated as solid dispersions as compared with their physical mixture and drug alone coun-terparts. Characterisation of the solid dispersions was performed using differential scanningcalorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and scanning electron mi-croscopy (SEM). DSC studies revealed that drugs were present in the amorphous form withinthe solid dispersions. FTIR spectra for the solid dispersions of drugs suggested that therewas a lack of interaction between PEG 8000 and the drug. However, the physical mixture ofphenacetin with PEG 8000 indicated the formation of hydrogen bond between phenacetin andthe carrier. Permeability of phenacetin and phenylbutazone was higher for solid dispersionsas compared with that of drug alone across Caco-2 cell monolayers. Permeability studies haveshown that both phenacetin and phenylbutazone, and their solid dispersions can be categorisedas well-absorbed compounds. © 2011 Wiley-Liss, Inc. and the American Pharmacists AssociationJ Pharm Sci 100:4281–4294, 2011Keywords: solid dispersions; PEG 8000; Phenacetin; Phenylbutazone; FTIR; DSC; Dissolu-tion studies; Permeability; amorphous form

INTRODUCTION

Poor solubility of drugs in water and gastrointestinalfluids leads to low dissolution rates and consequently,it can limit bioavailability. Bioavailability of a drug isdependent on its solubility and permeability.1 Tech-niques that have commonly been used to improvedissolution and bioavailability of poorly water-solubledrugs include salt formation,2 micronisation,3 the useof surfactants,4 particulate delivery systems5 and theformulation of solid dispersions.6 Solid dispersionsare defined as the dispersions of one or more activepharmaceutical ingredients in an inert hydrophilic

Correspondence to: Afzal R. Mohammed (Telephone: +44-121-2044183; Fax: +44-121-2044000; E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 100, 4281–4294 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

carrier in solid state. It is a widely used techniqueto enhance drug dissolution and in turn, it promotesbioavailability of insoluble drugs. Solid dispersionsin water-soluble carriers have attracted considerableinterest as a means of improving dissolution andbioavailability of a range of hydrophobic drugs.7 Theenhanced drug dissolution can be ascribed to multiplefactors, including conversion of crystalline drug intoamorphous form, reduction in particle size and an im-proved wettability due to inert hydrophilic carrier.8

The fabrication of solid dispersions is associatedwith the complexity of the release mechanism andthe multitude of factors that can affect it, includ-ing the properties of drug,9 particle size,10 proper-ties of the polymer forming the matrix,11 molecularweight,12 physical state13 and possible drug–polymerinteraction.14 Theories proposed for the drug releasemechanisms from solid dispersions are dependent onthe dissolution of both drug and polymer. Two widely

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studied mechanisms include: carrier-controlled re-lease with the release rate being influenced by thedissolution of the polymer and being entirely indepen-dent of the loaded drug,11,15 whereas there are otherdisperse systems that have shown release rate thatis mainly dependent on the properties of the drug.10

Although many papers have been published detailingdrug dissolution, few papers have reported examiningthe dissolution of the polymer. Currently, there is nomechanism that can predict the behaviour of a drugin solid dispersion as various factors are pivotal indictating drug release.

Caco-2 cell line has been widely used in in vitrosystem for predicting gastrointestinal absorption.16

According to Biopharmaceutics Classification Systemand US Food and Drug Administration, Caco-2 cellscan be used as a screening technique for new drug can-didates during drug discovery and development.17–19

The primary aim of the work presented was toset up a comprehensive and holistic investigation in-cluding formulation development of solid dispersionsof two water-insoluble model drugs, phenacetin andphenylbutazone, which have different physicochemi-cal properties. Phenacetin is an analgesic–antipyreticdrug20 having molecular weight 179.216 g/mol, melt-ing point 134C and logP 1.667. Phenylbutazone isan anti-inflammatory, antipyretic and analgesic drugwith a pKa of 4.5,21 melting point 105C, molecularweight 308.374 g/mol and logP 4.214. This study alsoassessed the release mechanisms by studying drug aswell as polymer dissolution and concluding the studywith an in vitro permeability investigation of the pre-pared formulations.

Polyethylene glycol 8000 (PEG 8000) was selectedas a carrier as it has excellent solubility in aque-ous medium and is nontoxic and nonimmunogenic.22

Solid dispersions containing different proportions ofPEG 8000 were prepared and characterised using dif-ferential scanning calorimetry (DSC), infrared spec-troscopy and scanning electron microscopy (SEM).Dissolution of the drug as well as the polymer (us-ing microviscometry) was investigated to determinethe release mechanism of the system.

MATERIALS AND METHODS

Materials

Phenacetin, phenylbutazone, phosphate bufferedsaline (PBS) tablets and potassium bromide werepurchased from Sigma–Aldrich, Dorset, UK. PEG8000 was obtained from Fluka (BioChemika, Hartle-pool, UK). High performance liquid chromatography(HPLC) grade water, acetonitrile, methanol, glacialacetic acid and absolute ethanol were obtained fromFisher Scientific, Leicestershire, UK.

Caco-2 cell line was purchased from AmericanType Culture Collection (ATCC), Middlesex, UK.Dulbecco’s modified eagle’s medium (DMEM), fe-tal bovine serum (FBS), nonessential amino acids(NEAA) and Hank’s balanced salt solution (HBSS)were purchased from Biosera, East Sussex, UK. Six-well transwell inserts were provided by Corning Inc,Life Sciences distributed by Appleton woods lim-ited, Birmingham, UK. Trypsin–ethylenediaminete-traacetic acid (1%), penicillin–streptomycin supple-mented with 2 mM glutamine (1%) and RNase freewater were purchased from Invitrogen, Paisley, UK.

Spectrophotometric Analysis

Drug concentration was measured and determinedin solution by spectrophotometric technique usinga quartz cuvette. Phenacetin and phenylbutazone(measured amounts) were dissolved in PBS solutionof pH 7.4, and Unicam ultraviolet (UV)–visible spec-trophotometer (200–400 nm) was used to determinethe wavelength of maximum absorption. A stock so-lution of phenacetin and phenylbutazone were pre-pared at 30:g/mL in PBS (pH 7.4) to carryout cal-ibration. The linearity of the calibration curve wasobtained in a concentration range of 2–14:g/mL andwas analysed by UV spectroscopy at λmax of 244 nm(phenacetin) and 236 nm (phenylbutazone).

Preparation of Solid Dispersion and Physical Mixture

Solid dispersions were prepared by melt fusion15 con-taining 5%, 10% and 15% (w/w) of phenacetin andphenylbutazone loading in PEG 8000. A homogeneoussolution was formed by heating the drug and the poly-mer at 120C on a heating plate for 15 min while stir-ring, until the drug dissolved completely in the melt.The solution was solidified under ambient conditionsafter pouring it into tablet moulds.

Phenacetin and phenylbutazone [5%, 10% and 15%(w/w)] and PEG 8000 were ground for 15 min in a mor-tar to make physical mixtures at room temperature(25C).

Drug Dissolution Studies

In vitro dissolution tests were performed to evalu-ate the dissolution of phenacetin, phenylbutazone,physical mixture and solid dispersions. The disso-lution studies were carried out using Hanson Re-search apparatus (SRII 6 Flask dissolution test sta-tion) fitted with a validate control unit. It was alsoequipped with 1-L round bottom flasks and basketsthat conform to the Apparatus 1 standards laid outin the United States Pharmacopeia.23 The dissolu-tion testing was carried out at a temperature of 37Cin 1000 mL of the dissolution media (PBS at a pHof 7.4) rotated at 100 rpm.15 At predetermined timepoints, 5 mL samples were taken, filtered (0.45:m)and analysed for drug content using UV–visible

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011 DOI 10.1002/jps

PHENACETIN AND PHENYLBUTAZONE SOLID DISPERSIONS WITH PEG 8000 4283

spectrophotometer and replaced with fresh 5 mL (pre-warmed to 37C) dissolution media after each sam-pling (all experiments were performed in triplicate).Experiments for dissolution studies were performedfor 60 min. The dissolution vessels were covered withlids to minimise and avoid evaporation.

Polymer Dissolution Studies

Microviscometry was used to measure the dissolutionof PEG 8000 from drug–PEG 8000 solid dispersions.The samples were measured on an AMVn version1.612047 microviscometer (Anton–Parr, Osterreich,Austria) equipped with the Visionlab software. Thefollowing conditions were used for each run sam-ple: temperature 25C, AMVn measuring programme(standard 50 × 4) and AMVn measuring system15084989.

Polyethylene glycol 8000 was dissolved in PBSover a concentration range of 2–10 mg/mL to pre-pare a calibration. The dissolution of PEG 8000 fromthe drug polymer mixed systems was measured us-ing microviscometry24 for all samples at all timeintervals. All the measurements were performed intriplicate.

Differential Scanning Calorimetry (DSC)

Diamond DSC (Perkin–Elmer) with a thermal an-alyzer, equipped with the Pyris software was em-ployed to obtain hyper-DSC data. Samples (2–5 mg)were crimped and placed on the sample furnace afterweighing into a nonhermetically sealed DSC samplepan. Heat flow rate of 500C/min was used to heatthe samples from 0C to 300C. Helium was used asa purge gas. In order to derive the melting points ofeach peak, onset temperature was measured. For ref-erence, an empty pan was crimped. All the measure-ments were performed in triplicate. Samples werealso heated at 10C/min from 0C to 300C using ni-trogen as the purging gas under normal DSC.

Infrared Spectroscopy

Fourier transform infrared spectroscopy (FTIR) spec-trometer from Pye Unicam Ltd. (Cambridge, UK) wasused to obtain the FTIR spectra. The samples weremixed thoroughly with potassium bromide at 1:100(sample: potassium bromide) weight ratio after beingground. A pressure of 5 tons was applied for 5 min tocompress the powder, in a hydraulic press, in orderto prepare potassium bromide discs. Scans were ob-tained at a resolution of 4 cm–1, from 4000 to 400 cm–1.All the studies were performed in triplicate.

Scanning Electron Microscopy (SEM)

Scanning electron microscopy data was achieved us-ing a scanning electron microscope, Stereoscan 90(Cambridge, UK). Double-sided adhesive carbon tapewas used to fix the sample powders to an aluminium

stub and it was made conductive for use in EmscopeSputter (SC 500) for 360 s at 10 mA by coating with adouble gold layer in vacuum (4 Psi). The samples werethen loaded into the SEM to obtain scanning electronmicrographs of the sample.

Solubility

For solubility studies, excess amount (known) of soliddispersion, physical mixture and drug alone wereadded in a 25-mL glass tube containing 10 mL PBS.The glass tubes were sealed and agitated at 120 rpmfor 24 h in a thermostated shaking water bath set at25C. The solutions were filtered through 0.45-:m fil-ter papers (Whatman), diluted and analysed by UVspectrophotometer at the corresponding wavelengthsfor each drug. The experiments were carried out intriplicate.

HPLC Analysis (Transport Studies)

High-performance liquid chromatography studieswere performed using a Dionex 1100 HPLC sys-tem with autosampler (AS50), gradient pump (GP50),detector (UVD170U) and a C18 analytical columnmaintained at 25C [ODS 3 column (Phenomenex),4.6:m × 150mm] with a particle size of 5:m. Cali-bration curves and samples were prepared in HBSS.The mobile phase consisted of acetonitrile (50%), dou-ble distilled water (50%) and 1 mL (per liter of mobilephase) of acetic acid. Phenacetin and phenylbutazonewere detected at a wavelength of 244 nm and 236 nm,at a flow rate of 1 mL/min. The injection volume wasmaintained at 20:L, and the retention time was2–4 min for phenacetin and 13–15 min for phenylbu-tazone.

Procedure for Caco-2 Cells Culture

Caco-2 cells with passage 70 were obtained fromATCC and were used at passages 90–100. Cellswere grown to 90% confluence in 75-cm2 T-flaskswith DMEM, supplemented with 10% FBS, 1% peni-cillin–streptomycin supplemented with 2 mM glu-tamine, and 1% NEAA. The culture medium waschanged every second day and cells were grown ata temperature of 37C and 5% CO2 with 95% relativehumidity. For the transport assay, cells were seededon top of six-well transwell culture plate (polycarbon-ate membrane) inserts (0.4:m pore size, 24 mm diam-eter and 4.7 cm2 surface or growth area) at a densityof 2 × 105cells/cm2. Transwell inserts were used byfirst adding the medium to the six-well plates, thenadding the transwell insert followed by the additionof the medium and cells to the inside compartmentof the transwell insert. Recommended six-transwellpermeable medium volume is 2.6 mL for plate welland 1.5 mL for the interior of transwell. An initialequilibrium period was used to improve the cell at-tachment by adding the medium to the six-well plate

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and then to the transwell insert. The plate was thenincubated for at least 1 h. The cells were then addedto fresh medium in the transwell insert and were re-turned to the incubator. The level was checked pe-riodically and fresh medium was added as required.The culture medium was replaced every 24–48 h. Theformation of tight junction of monolayers is deter-mined through the electrical resistance of the barrier.Thus, integrity of Caco-2 monolayers was measuredby transepithelial electrical resistance (TEER) to en-sure the formation of tight junctions using epithelialvoltohmmeter (EVOM). The TEER values were in therange of 500–600 Ω cm2 before and after the comple-tion of every study.

Permeability Studies

Caco-2 monolayers were used 21–25 days after seed-ing and cells cultured as submerged. The cells un-dergo complete differentiation after 21 days basedon and as evidenced by well-defined tight junctionthroughout the monolayers, as reported by previousresearchers.25,26 Apical to basolateral permeabilityof the drug and the solid dispersion was assessed.After 1 h of preincubation with drug-free transportmedium (HBSS), the medium containing the drugand the solid dispersion was introduced to the apicalside (1.80 mL). To determine the initial concentration(Co), a sample of 300:L was taken from the apicalside (1.50 mL remaining at the apical side). Samplealiquots (300:L) were taken from the basolateralside at given time intervals (0, 5, 10, 15, 20, 25, 30and 60 min). After each sampling, an equal volume offresh transport buffer (prewarmed at 37C) was addedto the receiver compartment (basal side), and the cellswere kept at a temperature of 37C and 5% CO2 with95% relative humidity during the experiment. Sam-ples were subsequently analysed by HPLC. All ex-periments were performed at 37C (n = 3). Apparentpermeability Papp (cm/s) was calculated according toEq. 1.

Papp = dQ/dt × 1/AC0 (1)

where dQ/dt is the rate of appearance of the drugson the basolateral side (nmol/s−1), Co is the initialconcentration on the apical side (mM), and A is thesurface area of the monolayer (cm2).

Statistical Analysis of Data

Student’s unpaired t-test with p < 0.05 was consid-ered significant. Statistical significance was calcu-lated by GraphPad prism software.

RESULTS AND DISCUSSION

Thermal Analysis (Differential Scanning Calorimetry)

Figures 1 and 2 show the hyper-DSC thermogramsof phenacetin, phenylbutazone, PEG 8000 and theirsolid dispersions. Hyper-DSC cycles were used to en-hance the thermal signal when compared with thestandard DSC thermograms. This also had the addi-tional benefit of minimising changes in morphologyand preventing any interactions during the heatingprocess. The thermograms of drug alone and polymeralone exhibited single endothermic peaks at around133.38C, 107.70C and 59.13C for phenacetin,phenylbutazone and PEG 8000, respectively. Inves-tigation of the heating scans of solid dispersions forphenacetin and phenylbutazone showed melting peakfor the polymer at around 59C with no endother-mic peak corresponding to the drug. The absence ofa peak at the temperature corresponding to the melt-ing of the drug could potentially be attributed to thesolubilisation and distribution of the drug within thehydrophilic polymer matrix resulting in the conver-sion of crystalline drug into amorphous form, whichwas reported in previous researches27,28 and in ourpapers.29,30

Differential scanning calorimetry scans for thephysical mixture revealed an interesting profile.Physical mixture of phenacetin with the polymer(Fig. 3) showed two transitions: the first correspond-ing to the melt of the polymer and the second due tothe melting of the drug. However, physical mixtureof phenylbutazone with the polymer (Fig. 4) resultedin the absence of melting endotherm correspondingto the drug as seen in the thermograms for the soliddispersion. To further investigate the binary physicalmixture of phenylbutazone, standard DSC at a scanrate of 10C/min was employed to gain an insight intoany transitions occurring during slow heating, in con-trast to the faster heating rate used in hyper-DSC.Analysis of samples containing 20%, 30% and 40%(w/w) drug content for the phenylbutazone showedonly a single peak corresponding to the melting ofthe polymer (Fig. 5). These results are in agreementwith the previous work reported on the thermal anal-ysis of physical mixtures of poorly soluble drugs andpolymers. Research published by Abdul-Fattah andBhargava31 and Ahuja et al.32 have shown that thephysical mixtures of the drug candidates resulted ina single peak corresponding to the polymer melt, withthe peak for the drug absent in the scans for the phys-ical mixture. The lack of the endotherm of the drughas been attributed to the melting and solubilisationof the drug within the molten carrier during the heat-ing of the sample. PEG 8000 is characterised by anonset melt temperature of around 59C, and the cor-responding endotherms for phenylbutazone occurs at107.70C. It may be possible that during the heating



Figure 1. Hyper-DSC thermograms consisting of 15% (w/w) solid dispersion of phenacetin,phenacetin alone and PEG 8000 alone. The arrow shows absence of drug endotherm for soliddispersion of phenacetin.

process for the analysis of thermogram for the phys-ical mixture, the molten carrier (which has nearlyhalf the melting temperature as compared with thedrug) begins to solubilise the drug, thereby dispers-ing it within its matrix with the consequence thatthe endotherm for the drug disappears completely.

In order to further investigate the differences in thethermal behaviour of the physical mixture (presenceand absence of drug endotherms), the samples weresubjected to infrared analysis to determine the possi-bility of any molecular interactions between the twoexcipients.

Figure 2. Hyper-DSC thermograms consisting of 15% (w/w) solid dispersion of phenylbuta-zone, phenylbutazone alone and PEG 8000 alone. The arrow shows absence of drug endothermfor solid dispersion of phenylbutazone.


4286 KHAN ET AL.

Figure 3. Hyper-DSC thermogram of physical mixture consisting of 15% (w/w) phenacetinand PEG 8000. The arrow shows presence of drug endotherm for physical mix of phenacetin.

Fourier Transform Infrared Spectroscopy (FTIR)

In order to characterise possible interactions betweenthe drug and the polymeric carrier, infrared spectrawere recorded. Four different spectra comprising ofdrug only, polymer only, physical mixture of the drugwith polymer and solid dispersion of the drug with thepolymer were recorded for each of the drugs investi-gated. The infrared spectra for the polymer was char-acterised by sharp peaks at 3450, 2891 and 1148 cm−1

corresponding to the stretching associated with O H,C H and C O bonds, respectively. Analysis of spectra

for phenylbutazone showed absorption bands at wavenumbers 1752, 1324 and 898 cm−1 corresponding tothe presence of C=O, C N aromatic amine and C H,respectively. Analysis of the spectra for the physicalmixture as well as the solid dispersion did not revealany changes in the specific absorption bands for boththe polymer as well as the drug, suggesting a lackof interaction between the two moieties (Fig. 6 andTable 1).

However, infrared spectra for the physical mix ofphenacetin showed broadening of peak at 3450 cm−1

associated with O H group in PEG 8000 and

Figure 4. Hyper-DSC thermogram of physical mixture consisting of 15% (w/w) phenylbuta-zone and PEG 8000. The arrow shows absence of drug endotherm for physical mix of phenylbu-tazone.



Figure 5. DSC thermograms of physical mixture consisting of 20%, 30% and 40% (w/w) (bot-tom to top) phenylbutazone and PEG 8000.

Figure 6. Fourier transform infrared spectra of (bottom to top) (a) PEG 8000, (b) phenylbu-tazone, (c) physical mixture of PEG 8000-phenylbutazone and (d) solid dispersion of PEG 8000and phenylbutazone.


4288 KHAN ET AL.

Table 1. Shows Wave Number (cm−1) and Functional Groups for PEG 8000, Phenylbutazone, Physical Mixture andSolid Dispersion of Phenylbutazone Using FTIR

PEG 8000 Phenylbutazone AlonePhenylbutazone Physical

MixPhenylbutazone Solid

Dispersion

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

1148 C O 693 C H 693 C H 693 C H1467 C H 753 C H 753 C H 754 C H2891 C H 898 C H 898 C H 898 C H3450 O H 1324 C N 1148 C O 1148 C O

1752 C=O 1320 C N 1318 C N2956 C H 1467 C H 1467 C H

1753 C=O 1752 C=O2891 C H 2891 C H2955 C H 2956 C H3450 O H 3450 O H

3286 cm−1 associated with N H amine group inphenacetin, suggesting the formation of hydrogenbond between the two groups. The broadening of thepeak (3450 cm−1 for PEG associated with O H) sug-gests the formation of hydrogen bond between thelone pair of electrons in the nitrogen atom and thehydrogen atom (Fig. 7 and Table 2). Hydrogen bond-ing was reported for physical mixture by Rawlinsonet al.33 and Hendriksen et al.34 investigating the crys-tallisation of paracetamol in the presence of struc-turally related compounds. The formation of hydro-gen bond between phenacetin and the polymer evenas a physical mixture could possibly explain the DSCscans reported above. It may be possible that lowertemperature fails to break the hydrogen bonding as-sociation between the drug and the polymer wherebythe mixing of the two components during the heat-

ing process of the DSC run is impeded, as a result ofwhich two distinct peaks corresponding to the meltof the polymer and the drug were obtained. However,the formulation of solid dispersion of the drug whichis characterised by continuous heating and the sup-plementation of the process with continuous stirringwould explain the lack of peak in the DSC scans char-acteristic of the drug, suggesting the drug to be in adispersed amorphous state.

Drug Dissolution Studies

The solid dispersions of phenacetin and phenylbu-tazone studied showed that drugs released faster insolid dispersion as compared with drug alone and itsphysical mixture (Figs. 8 and 9). Solid dispersionsof many poorly water-soluble drugs with hydrophiliccarrier matrix have been formulated for improving

Figure 7. Fourier transform infrared spectra of (bottom to top) (a) PEG 8000, (b) phenacetin,(c) physical mixture of PEG 8000-phenacetin and (d) solid dispersion of PEG 8000 andphenacetin.



Table 2. Shows Wave Number (cm−1) and Functional Groups for PEG 8000, phenacetin, Physical Mixture and SolidDispersion of Phenacetin Using FTIR

PEG 8000 Phenacetin Phenacetin Physical Mix Phenacetin Solid Dispersion

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

Wave No(cm−1)

FunctionalGroup

1148 C O 838 C H 840 C H 839 C H1467 C H 1267 C N 1150 C O 1149 C O2891 C H 2928 C H 1258 C N 1263 C N3450 O H 3286 N H 1468 C H 1467 C H

2889 C H 2888 C H2935 C H 2930 C H3340 N H 3289 N H3470 O H 3452 O H

drug dissolution rate.35 Drugs released faster in5% (w/w) solid dispersion as compared with 10%and 15% (w/w) solid dispersions. The drug releasedfrom 5% (w/w) solid dispersion of phenylbutazone was100% as compared with 10% and 15% (w/w) soliddispersion over the entire length of the time period[in case of 10% (w/w), drug release was 84% and for15% (w/w), drug release was 73%]. The drug releasewas slower from the dispersions containing high drugcontent as compared with that from low drug con-tent formulations. The study of phenacetin for 5%,10% and 15% (w/w) formulations showed a similartrend. Lin and Cham36 have reported that solid dis-persions in PEG 6000 of naproxen displayed fasterdrug release from 5% or 10% (w/w) naproxen load-ing as compared with 20%, 30% or 50% (w/w) drugloading. The importance of carrier in the solid dis-persion was demonstrated in a study of 14 differentdrugs in PEG 6000.15 Dubois and Ford15 reported thatfor a certain drug present in the formulation in lowdrug to carrier ratio, the release rate is carrier con-trolled irrespective of the drug properties. Further-more, the bioavailability of poorly soluble drugs maybe enhanced by formulating solid dispersions result-ing in an increase in the drug dissolution rate andtheir saturation solubility in the gastrointestinal flu-ids. Solid dispersion is regarded as a potential meansof improving the dissolution behaviour of poorly solu-ble drugs, as it was previously presented.11,37,38 Chiouand Riegelman39 demonstrated an increase in the re-lease rate of griseofulvin in PEG solid dispersions.Similar results were obtained in other studies in-vestigating insoluble drugs, including ketoprofen,40

oxazepam,41 carbamazepine42 and zolpidem.43

It is well established that PEG may increase thesolubility of drugs, particularly at high concentra-tions. These results also reflect that the dissolutionrate is also influenced by drug concentration in thesolid dispersion with lower drug content, resulting inbetter improvement in the dissolution rate. The drugto carrier ratio in a solid dispersion is one of the deter-mining factors in the performance of solid dispersion.

Figure 8. Dissolution profiles of phenacetin/PEG 8000solid dispersion; 5% (w/w), 10% (w/w), 15% (w/w), 15%(w/w) physical mix and 15% (w/w) phenacetin alone ispresented for comparison. Data are expressed as mean[n = 3; mean ± standard deviation (SD)].

Figure 9. Dissolution profiles of phenylbutazone/PEG8000 solid dispersion; 5% (w/w), 10% (w/w), 15% (w/w), 15%(w/w) physical mix and 15% (w/w) phenylbutazone aloneis presented for comparison. Data are expressed as mean(n = 3; mean ± SD).

This could possibly be attributed to the wettability of-fered by the polymer and the conversion of crystallinedrug into amorphous form. The high percentage ofthe polymer can promote better wettability with animproved solubility due to the complete dispersion


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Figure 10. Dissolution of PEG 8000 and phenacetin from5%, 10% and 15% (w/w) solid dispersion. Data are expressedas mean (n = 3; mean ± SD).

of the drug in the solid dispersion, resulting in highdrug release. Identical findings were demonstratedfor lorazepam–PEG solid dispersion, where a linearrelation was found for polymer weight ratio and thedissolution rate constant.44

Polymer Dissolution Studies

Microviscometry was used to measure dissolution ofthe polymer at predetermined time intervals. Thetrend that solid dispersion system of phenacetin andphenylbutazone [5%, 10% and 15% (w/w)] follow forthe dissolution of PEG 8000 and drug is illustratedin Figures 10 and 11. The polymer and drug releasefrom 5% (w/w) solid dispersion of phenylbutazone was85% and 54% in 30 min for the polymer and the drug,respectively. On the contrary, 10% (w/w) formulationof phenylbutazone showed a release rate of 75% and38% for the polymer and the drug, respectively. How-ever, for the same time interval, the percent releasewas 71% for the polymer and 33% for the drug in 15%(w/w) solid dispersion of phenylbutazone.

The percent release from 5% (w/w) solid disper-sion of phenacetin was 21% and 4% for PEG 8000and phenacetin, respectively, after the first 5 min.The polymer released was 100% over a time lengthof 30 min as compared with 76% of drug release. Therelease profile for 10% (w/w) phenacetin dispersion,

Figure 11. Dissolution of PEG 8000 and phenylbutazonefrom 5%, 10% and 15% (w/w) solid dispersion. Data areexpressed as mean (n = 3; mean ± SD).

6% for polymer and 3% for drug was observed at5 min. The polymer release was 100% over the entirelength of time period (60 min) and the drug releasewas 63%. The dissolution profile of 15% (w/w) soliddispersion revealed identical release patterns as thepercent drug released was 12% and the polymer was8% after 5 min. However, the release increased to 29%for the drug and 100% for the polymer after 60 mintime interval.

It can be postulated that various mechanisms de-termining the dissolution prevail at different ratiosof polymer and drug for different drugs. This studyenables us to suggest, through measuring the disso-lution of the polymer and the drug simultaneously,that polymer dissolution is the vital mechanism whichcontrols the dissolution of phenylbutazone. Hence thedrug release is ruled by dissolution of the polymer.Phenacetin formulation demonstrates an interestingprofile: In case of 5% (w/w) solid dispersion, the dis-solution of drug is controlled by the drug and theamount present in the formulation. In case of 10%(w/w) and 15% (w/w) solid dispersions, drug releasewas controlled by polymer dissolution. Thus, it canrightly be deduced that by altering the physical prop-erties of the drug (crystalline form into amorphousform) and the polymer concentrations, the dissolutionrate can be altered, suggesting the need for optimi-sation of polymer concentration during formulationdevelopment.

Shape and Surface Morphology (Scanning ElectronMicroscopy)

Scanning electron microscopy at different magnifi-cations was used for investigating the morphologi-cal differences of samples (drugs, PEG 8000, physi-cal mixtures and formulations). Figure 12 shows thatphenacetin consists of rod shaped crystals, phenylbu-tazone is made up of needle shaped crystals45 andPEG 8000 exists in irregular crystalline shapes. Thephysical mixture of phenacetin and phenylbutazonewith the carrier showed that the drug is in the crys-talline form as is evidenced by the presence of drugattached on the surface of carrier (not dispersedin the carrier completely), which is justified by theDSC results (see above) of physical mixtures. On theother hand, the photomicrographs of the solid dis-persion show that drugs were dispersed in the car-rier. It is clear that morphological differences wereseen between the two drugs and their physical mix-tures and solid dispersion counterparts. Drug mustbe dispersed/soluble/reduced in particle size withinthe solid dispersions without crystal formation in or-der to enhance the dissolution profile.44 These obser-vations also show that the determinations from theDSC study are tenable. SEM studies further sug-gested that the surface properties of all drugs andPEG 8000 were lost during the formation of solid



Figure 12. Scanning electron microphotographs of (a) phenacetin (219×), (b) phenylbutazone(173×), (c) physical mixture of phenacetin-PEG 8000 binary system (238×), (d) physical mixtureof phenylbutazone-PEG 8000 binary system (189×), (e) solid dispersion of phenacetin-PEG 8000binary systems (379×), (f) solid dispersion of phenylbutazone-PEG 8000 binary system (156×)and (h) PEG 8000 (178×).

dispersion system by melting and solidification, re-sulting in the dispersion of the drug molecules withinthe carrier matrix. Moreover, these results also sub-stantiate an enhancement in the dissolution profileof the drug candidates, possibly due to dispersion ofthe drug molecules and the absence of any crystallineparticles characterised by DSC.

Solubility Studies

The solubility of phenacetin and phenylbutazone inPBS (pH 7.4) from their respective solid dispersions,physical mixtures and drug alone are tabulated inTable 3. The solubility of phenylbutazone alone wasfound to be 6.34 ± 0.68:g/mL, which was lower thanthat for the physical mixture (9.97 ± 1.24:g/mL). Thesolubility value for phenylbutazone dispersion was44.68 ± 2.7:g/mL, which is significantly higher (sev-enfold) than the drug alone. Similar trend was alsoobserved for solubility analysis of phenacetin. Thesolubility in case of phenacetin was found to haveincreased (ten times) as compared with that of drug

alone. Unpaired t-test results using GraphPad soft-ware showed that the difference was statistically sig-nificant for both formulations when compared withfree drug or physical mix (p < 0.001 for phenacetinsolid dispersion when compared with drug alone orphysical mix; p < 0.001 for phenylbutazone solid dis-persion when compared with drug alone and p < 0.001for phenylbutazone solid dispersion when comparedwith physical mix). The increase in the solubility inphysical mixtures can be attributed to the wettabil-ity of drugs or conversion of crystalline to amorphousform.46 The study revealed that saturation drug sol-ubility was greater in the solid dispersions, followedby physical mixtures and drug alone.

Permeability Studies

Caco-2 cell monolayers have been recognised asin vitro model membranes and are vital for the rapidscreening of intestinal drug absorption. The cumu-lative percent transfer of phenacetin and phenylbu-tazone from apical to basal (:g/2.5mL) across


4292 KHAN ET AL.

Table 3. Solubility of Solid Dispersions, Physical Mixtures and Drug Alone in Phosphate Buffer Saline

Drugs Solid Dispersions (:g/mL) Physical Mixtures (:g/mL) Drug Alone (:g/mL)

Phenacetin 69.44 ± 2.27 10.47 ± 0.63 7.42 ± 0.56Phenylbutazone 44.68 ± 2.7 9.97 ± 1.24 6.34 ± 0.68

Data are expressed as mean (n = 3; mean ± SD).

Figure 13. Apical-to-basal permeability of solid disper-sion of phenacetin () and phenacetin alone () acrossCaco-2 monolayers. Data are expressed as mean (n = 3;mean ± SD).

Caco-2 monolayer at different time points forphenacetin alone and its solid dispersion are shownin Figures 13 and 14. The transport of phenacetinand phenylbutazone through Caco-2 monolayers wasmore from solid dispersions for both the drugs.Phenacetin permeability from solid dispersion after60 min was 53:g/2.5mL as compared with 33:g/2.5mL for drug alone, and the difference was found tobe statistically insignificant (p > 0.1 for phenacetinsolid dispersion when compared with drug alone).Phenylbutazone permeability from solid dispersionafter 60 min was 13:g/2.5mL as compared with 9:g/2.5mL for drug alone, and the difference was found tobe statistically significant (p < 0.0001 for phenylbu-tazone solid dispersion when compared with drugalone). The apparent permeability coefficients forphenacetin, phenylbutazone and their solid disper-sion were measured at predetermined time intervalsfor the solid dispersions and drug alone, respectively,as tabulated in Table 4.

Permeability coefficient (Papp) is classified as lowPapp (<1 × 10−6 cm/s), moderate Papp (=1–10 ×10−6 cm/s) or high Papp (>10 × 10−6 cm/s) corre-sponding to poor, moderate and well-absorbed com-pounds, respectively.47 This study demonstrates that

Figure 14. Apical-to-basal permeability of solid disper-sion of phenylbutazone () and phenylbutazone alone ()across Caco-2 monolayers. Data are expressed as mean (n= 3; mean ± SD).

phenacetin, phenylbutazone and their solid disper-sions can be categorised as well-absorbed compounds.The pattern of absorption followed the in vitro disso-lution behaviour of the solid dispersion reinforcingthe role of PEG 8000 as a solubiliser for the drugin formulation (conversion of crystalline drug intoamorphous form have resulted in an increase in sol-ubility). Recent studies have shown that some excip-ients, which are commonly added to different phar-maceutical formulations, could inhibit the function ofphospho-glycoprotein (P-gp) in the intestine. Thesedifferent excipients, which are added to formulations,are considered to be nontoxic and inert. To achieve anincrease in drug transport and permeability by P-gpinhibition, the presence of polyoxyethylene groups isrequired.48 The possible explanation for the increasein permeability of the drug may be of different factorssuch as conversion of crystalline form into amorphousform, altering the dissolution rate, presence of carrierand an inhibition of efflux systems.29,30 The solubil-ising effect of PEG 8000 allows the drug to quicklyassociate into the aqueous surrounding, possibly bymolecular complexation. This enables the solubiliseddrug to be absorbed rapidly as compared with thedrug without any carrier.

Table 4. Shows the Permeability Coefficients (Papp) Value Calculated from Solid Dispersion and Drug Alone

DrugsPermeability Coefficients Papp × 10−6 (cm/s)

(Solid Dispersion)Permeability Coefficients Papp × 10−6 (cm/s)

(Drug Alone)

Phenacetin 42.52 ± 5.97 29.37 ± 4.12Phenylbutazone 15.98 ± 0.99 10.04 ± 0.30

Data are expressed as mean (n = 7; mean ± S.D.)



CONCLUSIONS

Phenacetin and phenylbutazone were converted toamorphous form during the manufacture of solid dis-persions, which in turn enhanced the dissolution ofthe drug. FTIR analyses of solid dispersion of thedrugs suggested that there was a lack of interac-tion between PEG 8000 and drugs. However, physicalmixture of phenacetin with PEG 8000 indicated theformation of hydrogen bond between the phenacetinand PEG 8000. This study also demonstrated thatpolymer dissolution is the vital mechanism, whichcontrols the dissolution of the drug. It can be con-cluded that alteration of the physical properties ofthe drug (conversion to amorphous form) and poly-mer concentrations can affect the dissolution rate ofthe drug. This study demonstrated that phenacetin,phenylbutazone and formulations can be categorisedas well-absorbed compounds, and permeability washigher from solid dispersions as compared with freedrug.

ACKNOWLEDGMENTS

The authors would like to acknowledge Aston Uni-versity (Overseas bursary scholarship) for partiallyfunding this research studentship.

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