Materials Science and Engineering C 33 (2013) 596–602

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Materials Science and Engineering C

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Compatibility studies of nevirapine in physical mixtures with excipients fororal HAART

G.G.G. de Oliveira a, H.G. Ferraz a, P. Severino b,c, E.B. Souto c,d,⁎a Department of Pharmacy, Faculty of Pharmaceutical Health, University of São Paulo, São Paulo 05508-900, Brazilb Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas, Campinas 13083-970, Brazilc Department of Pharmaceutical Technology, Faculty of Health Sciences, Fernando Pessoa University, Porto 4200-150, Portugald Institute for Biotechnology and Bioengineering, Centre for Genomics and Biotechnology, University of Trás-os-Montes e Alto Douro (IBB-CGB/UTAD), 5001-801 Vila Real, Portugal

⁎ Corresponding author at: Faculty of Health Sciences, FCarlos daMaia 296, Office S.1, P-4200-150 Porto, Portugalfax: +351 22 550 4637.

E-mail address: eliana@ufp.edu.pt (E.B. Souto).

0928-4931/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.msec.2012.09.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 August 2011Received in revised form 30 August 2012Accepted 30 September 2012Available online 6 October 2012

Keywords:NevirapineDifferential scanning calorimetry (DSC)Thermogravimetric analysis (TGA)SolubilityIntrinsic dissolutionPhotostability

Nevirapine is a hydrophobic non-nucleoside reverse transcriptase inhibitor, used in first line regimens ofhighly active antiretroviral therapy (HAART). The drug has more than one crystalline form, which mayhave implications for its behaviour during production and also for its in vivo performance. This study wasaimed at exploring the suitability of thermoanalytical methods for the solid-state characterization of commercialcrystalline forms of nevirapine. The drug powderwas characterized by ultraviolet spectrophotometry, stereoscopy,scanning electron microscopy, wide-angle X-ray diffraction, measurements of density, flowability, solubility andintrinsic dissolution rate (IDR), differential scanning calorimetry, thermogravimetric analysis, and photostabilitymeasurements. The results showed that nevirapine has high stability and is not susceptible to degradationunder light exposure. The drug showed compatibility with the excipients tested (lactose, microcrystalline cellu-lose, polyvinylpyrrolidone and polyvinyl acetate copolymer (PVP/PVA), and hydroxypropylmethylcellulose(HPMC)). Nevirapine has low solubility, an acidmedium being themost appropriate medium for assessing the re-lease of the drug from dosage forms. However, the data obtained from IDR testing indicate that dissolution is thecritical factor for the bioavailability of this drug.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Thedevelopment of solid dosage forms for the purpose of oral admin-istration of antiretroviral drugs to treat HIV infection by highly active an-tiretroviral therapy (HAART) is becoming increasingly common, sincetheHIV epidemic is still infecting a large number of individuals annually.Knowledge about novel pharmaceutical dosage forms is of paramountimportance in this context [1–3]. Nevirapine is a hydrophobic non-nucleoside reverse transcriptase inhibitor, used in first line regimens ofantiretroviral therapy [4,5]. The drug exists in more than one crystallineform, namely the hemiethanolate (form I), hemiacetonitrilate (form II),hemichloroformate (form III), hemi-THF solvate (form IV), mixedhemiethanolate hemihydrate (form V) and hemitoluenate (form VI),reflecting physicochemical changes during production, and this also af-fects the in vivo performance upon administration [6].

Because of the special properties of nevirapine, pre-formulationstudies are required to test the compatibility between the drug and

ernando Pessoa University, Rua. Tel.: +351 22 507 4630x3056;

rights reserved.

excipients. Incompatibilities may lead to an accelerated loss of power,complexation, acid–base interaction or the formation of a eutectic mix-ture, resulting in low bioavailability and lack of stability [7,8].

In vitro dissolution testing is the most important method in the de-velopment of solid dosage forms [9–13]. It can be used to predict the per-formance of a dosage form, especially when drug release is the limitingfactor in the absorption process [12,14]. The Biopharmaceutical Classifi-cation System (BCS) [15,16] is a tool that can be used to design a dissolu-tion testing programme for predicting the dissolution rate, solubility andintestinal permeability. These parameters affect the in vivo performanceof the drug, and therefore the pharmacological activity [17]. Examples ofdrugs that have been tested in this way, such as ayurvedic medicinalherbs [18], nateglinide [19], ketoprofen [20], benznidazole [9] andlercanidipine [21], have been reported in the literature.

The aim of the present study was to evaluate the physicochemicalcharacteristics of nevirapine relevant to further processing into solid dos-age forms. The physicochemical properties of the drug powder werecharacterized by ultraviolet (UV) spectrophotometry, stereoscopy, scan-ning electron microscopy (SEM), wide-angle X-ray diffraction (WAXD),differential scanning calorimetry (DSC), thermogravimetric analysis(TGA), infrared (IR) spectroscopy and photostability measurements.Measurements of the density, flowability, solubility and intrinsic dis-solution rate (IDR) were also carried out on the bulk material.

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2. Material and methods

2.1. Material

Nevirapine was provided by Cristália (Itapira, Brazil). Thepolyvinylpyrrolidone copolymer/polyvinyl acetate (PVP/PVA) de-rivatives Kollidon® VA64 (vinylpyrrolidone–vinyl acetate copoly-mer, (C6H9NO)n×(C4H6O2)m) and Kollidon® K30 (ethenyl-2-pyrrolidinone homopolymer, (C6H9NO)n) were donated by BASF(São Paulo, Brazil). Hydroxypropylmethylcellulose (HPMC) (Methocel®K4M) was obtained from Dow Chemical Company (São Paulo, Brazil),microcrystalline cellulose PH 101 was purchased from BlanverFarmoquímica (Taboão da Serra, Brazil) and lactose was purchasedfrom Pharmatose (São Paulo, Brazil).

2.2. Methods

2.2.1. Quantitative analysis of nevirapineThe nevirapine was quantified by UV spectrophotometry (DU-640,

Beckman Coulter, Fullerton, CA, USA); it was analysed at 294 nmagainst a calibration curve (R2=0.999), employing solutions of second-ary standards in various buffers with pH values ranging from 1.2 to 7.0.

2.2.2. Stereoscopy and scanning electron microscopyThe appearance of the solid bulk powder of the drug was analysed

in a stereoscope (Motic SMZ® 168 T, coupled to a Fujifilm FinepixS5100®digital camera) at amagnification of 10–120×. The data obtainedrelated to the colour and appearance of the solidmaterial and the particleshape. Micrographs of nevirapine samples were obtained using a scan-ning electronmicroscope (LEO,model LEO440i, NewYork, USA). The sur-faces of the samples were metallized with a gold coating using a sputtercoater (Polaron, model SC 7620, Schwalbach, Germany) prior to record-ing the images.

2.2.3. Wide-angle X-ray diffractionWAXS analysis (Rigaku Ultima X-ray diffractometer, TheWoodlands,

TX, USA) was performed using monochromatic 1.5418 Å radiation froma tubewith a copper target operated at 40 kV and 30 mA (1.2 kW), and ascintillation detector. The samples were fixed with gel-type amorphousHIVAC-G Shin-Etsu silicone. The pitch angle for each sample was 0.05°;the irradiation timewas 5 s,with rotation at 30 rpm. The interlayer spac-ings dwere calculated from the reflections using Bragg's equation as fol-lows:

d ¼ λsin2θ

where λ is the wavelength of the incident X-ray beam and θ is the scat-tering angle. The parameter d is the separation between the planes be-longing to a particular set of planes in the crystal lattice structure.

2.2.4. Determination of densityAccurately weighed 10 g amounts of nevirapine were sieved

(mesh 20), and then transferred to beakers for analysis on a standardtap density tester (TAP-2, Logan Instruments Corporation, Somerset,NJ, USA). The parameters used for the tap density test were 2000 tapsover a period of 10 min, which was found to be the minimum requiredto stabilize the final volume. The initial volume (Vi) was recorded,followed by measurements after 1, 2, 5 and 10 min. These measure-ments were used to calculate the final volume (Vf). The apparent densi-ty (dap) and compacted density (dcp) were then calculated from thefollowing equations:

dap ¼ Mi=V i

dcp ¼ Mi=V f

where dap is in g/cm3, Vi is in cm3,Mi is the initial mass (in g), dcp is ing/cm3 and Vf is in cm3.

Determination of the true density was performed using a heliumpycnometer (Quantacrome model 1000, Boynton Beach, FL, USA). Ac-curately weighted 1.0 g samples of nevirapine were analysed using asmall crucible, after a dehumidifying flow of helium had been appliedfor 3 min. Five readings were taken, with a variation of less than0.01% standard error. The true density (ρ) was calculated as the ratioof the mass (m) to the volume (V), using the following equation:

ρ ¼ mV:

2.2.5. FlowabilityTo assess the flowability of mixtures of nevirapine and excipients

as used in pellets, the compressibility index (CpI), the Carr indexand the Hausner ratio were determined as follows:

CpI ¼ dcp−dapdcp

!

Carr index ¼ dcp−dapdcp

!� 100

Hausner ratio ¼ dcpdap

where dap is the apparent density (g/cm3) and dcp is the compacteddensity (g/cm3).

2.2.6. SolubilityTo determine themaximumsolubility of nevirapine, an excess of the

drug (~250 mg) was transferred to vials containing a series of aqueoussolutions of hydrochloric acid (from 0.1 to 0.01 M) and 0.1 M phos-phate buffer solutions (at varying pH from 1.2 to 7.2) with stirring at200 rpm for 72 h, at a constant temperature of 37 °C in a shaker(Tecnal, model TE-420, Piracicaba, Brazil). The samples were thencentrifuged and quantified by spectrophotometry at 294 nm. The ex-periments were carried out in triplicate.

2.2.7. Effect of surfactant on solubilityTo study the impact of surfactants on the solubility of nevirapine,

tests were carried out in phosphate buffers in the presence of the an-ionic surfactant sodium lauryl sulphate at concentrations of 0.1, 0.5and 1.0% (w/w). This procedure was used because there was interfer-ence between the absorption of the excipients and the absorption ofthe drug at a wavelength of 294 nm.

2.2.8. Intrinsic dissolution rateTo determine the intrinsic dissolution rate, a device designed by us

was coupled to a conventional dissolution test apparatus. Samples of ne-virapine (200 mg) were transferred to the test apparatus by subjectingthem to compression (1000 kg/cm2) in a hydraulic press (equippedwith an American Lab WIKA pressure gauge, Iperó, Brazil) for 1 min.The dissolution test apparatus (Logan Instruments, model D-800,Somerset, NJ, USA) was located at 1.5 cm from the bottom of a tank.The tank had a volume of 900 mL, and was filled with 0.1 N HCl at atemperature of 37 °C. The experiments were performed in triplicatefor each condition, using a modified Wood apparatus (Varian Inc.,Vankel Division, Cary, NC, USA).

2.2.9. Differential scanning calorimetryCharacterization of nevirapine and of physical mixtures of nevira-

pine and excipients was performed by thermal analysis in a TA-2920apparatus (TA Instruments, New Castle, DE, USA) using a temperature

Fig. 1. Bulk nevirapine powder analysed by stereoscopy with (a) 60× and (b) 120×, and by scanning electron microscopy at (c) 1000× and (d) 3000×.

598 G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602

range from 25 to 300 °C. A sample mass of about 3.0 mg was weighedin a tightly closed aluminium container, and then analysed at a heatingrate of 10 °C/min under a dynamic atmosphere of nitrogen with a flowrate of 50 mL/min. The thermodynamic parameters were analysedusing the Universal Analysis® programme from TA.

0

1000

2000

3000

4000

0 10 20 30 40 50

Lin

(cp

s)

2 theta

Fig. 2. WAXD diffractogram of neviparine.

0.00

0.20

0.40

0.60

0.80

0

4

8

12

16

20

0 1 2 5 10 15

Com

pact

des

nsit

y (g

/cm

3 )

Time (min)

Vol

ume

(cm

3 )

Fig. 3. Volume (columns) and compact density (line) versus time analysed during15 min.

2.2.10. Thermogravimetric analysisCharacterization of nevirapine and of physical mixtures of nevira-

pine and excipients was performed by thermogravimetry using athermoanalytical balance (TA-2950, TA Instruments, New Castle, DE,USA) over a temperature range from 25 to 600 °C. A platinum cruciblecontaining a sample mass of 10.0 mg was used at a heating rate of10 °C/min under a dynamic nitrogen atmosphere with a flow rate of100 mL/min. The data were analysed using the Universal Analysis®programme.

2.2.11. PhotostabilityThe stability of nevirapine under light exposure was evaluated in a

photostability chamber (Nova Ética, Farma A 424C, São Paulo, Brazil)[22–24]. Nevirapine, in the form of the bulk material and standard20 μg/mL solutions in several different buffers, was subjected tolight exposure for 24 h. This period was chosen after calibration ofthe equipment according to the actinometrical method. This methodis based on the quantification of a solution of 2% (w/v) quininemonohydrochloride before exposure (at t=0) and at predeterminedtime intervals until 0.9 absorbance is reached. The degradation of thenevirapine was checked following UV spectrophotometry, DSC andTGA.

Table 1Data obtained with the helium pycnometer equipment related to the different readingsof a sample of nevirapine (m=0.2207 g).

Readings Volume True density

1 0.1174 1.88062 0.1362 1.80593 0.1222 1.62014 0.1376 1.60415 0.1357 1.6268Average 0.1365 cm3

True density 1.6170 g/cm3±0.0008%

0

1

2

3

4

5

6

7

8

0

1

2

3

4

5

6

HCl 0.1 N HCl 0.01 N Buffer pH 4.5Buffer pH 6.0Buffer pH 6.8 water Buffer pH 7.2

Buf

fer

pH

Solu

bilit

y (g

/L)

Fig. 4. Values of solubility of nevirapine (g/L) in various buffer solutions. The pH valuesof the media in which the tests were conducted are also presented.

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3. Results and discussion

Using stereoscopy to analyse the bulk powder, the nevirapineappeared to be a partially crystalline material (Fig. 1). When themagnification was increased, more detail was seen and elongatedparticles were observed. Using SEM analysis, further details of a con-tinuous mass of powder were obtained, consistent with a crystallinestructure. Similar results were obtained by Chadha et al. [6] andMohammed et al. [25] in analyses of commercial nevirapine, wherethe crystalline form of the drug was not clearly defined.

To confirm the crystallinity of the nevirapine powder, WAXD anal-ysis was carried out (Fig. 2). Interlayer spacings d of 9.2695, 13.3381and 25.2979 Å were observed. These results characterize form I of ne-virapine [26].

Flowability is an important factor in several processes in the phar-maceutical industry, including the compression process. Fig. 3 showsthe results for the apparent and compacted density. A reduction in thevolume of the powder was observed soon after the start of the test, andthe volume stabilized after 5 min. The use of 2000 taps is usuallyrecommended for measurements of the compacted density; this num-ber was obtained by checking for changes in volume during tests untilthere was stabilization of the observed volume [27]. The result for thedensity of the compacted powder in our tests was 0.5748 g/cm3.

The compressibility index CpI is related to the accommodation of theparticles [28]. The result for the Hausner ratio was 1.45, and that for theCarr index was 31.24%. A Hausner ratio below 1.25 indicates good com-pressibility. The Carr index may be indicative of the flowability and de-gree of packing of a material, which are relevant properties when thematrices of a tableting machine are being filled. A Carr index lowerthan 15% indicates an adequate flow of the powder and stable packing,whereas values above 25% are characteristic of poor flowproperties [29].

NN

N

CH3

NH

O

+ H 2 O

Fig. 5. Diagram representing the balance of molecular and

To improve flowability, changes in the crystal structure or in the particlesize have been suggested, these being criteria for comparison betweendifferent batches of raw material.

The data obtained with the helium pycnometer are shown inTable 1. The volume and true density obtained for the nevirapinewere 0.1365 cm3±0.009329 and 1.6170 g/cm3±0.0008%, respec-tively. There is a significant difference between this value for thedensity and the data presented earlier for the compacted density ofthe same material. The difference between the two data sets is relatedto how they were obtained. The compacted density takes account ofthe total volume occupied by the powder. Although the particles arein contact, however, empty spaces still exist between the particleseven after the compaction operation. The data from the pycnometerare obtained from the difference between the volumes occupied bythe inert gas (He) in the crucible container with and without the pow-der. The empty space in the powder is occupied by the gas, and the truedensity of the drug is determined from the difference in volume.

Solubility is an important parameter both for the in vitro evalua-tion of solid dosage forms and for proper dissolution and subsequentabsorption in vivo. According to the BCS, for a drug to be consideredhighly soluble, a dose/solubility value of less than 250 mL is necessaryover the whole pH range to which the drug will be exposed in the gas-trointestinal tract. Data obtained, after equilibration, for the solubility ofthe drug in various buffermedia are shown in Fig. 4. According to the sol-ubility data and based on the usual dose of the drug (200 mg), it can beestimated that only the conditions present in the stomach (pHb2)will be sufficient for the complete dissolution of nevirapine. Thedose/solubility value in other media is greater than 250 mL. Accordingto the BCS, this value defines the drug as poorly soluble, and, possibly,the step of dissolution of the drug from the dosage form may interferewith its bioavailability [30,31].

From Fig. 5, it can be seen that nevirapine is sensitive to pH, andthis is one of the factors contributing to its dissolution. In an acidmediumcontaining >2.10 g/L [H3O+], the solubility of the drug was 4.99 g/L.When the environment was changed using solutions with different pHvalues, the solubility of the drugwas reduced by a factor of 10–40. Struc-tural changes in the drugmolecule in solution are responsible for the dif-ferent solubility values at different pH values. The electron donor sites(nucleophiles) in nevirapine structure, represented by the pyridine ni-trogen atoms, can be protonated under acidic conditions, forming a sta-ble zwitterion form. By reducing the concentration of H3O+ ions, thisform tends to stabilize the molecule [32].

Aungst et al. [33] studied an antiretroviral drug (DPC 961) of lowsolubility and high permeability in vitro; the dissolution was clearlyimpaired in media with pH values from 4.5 to 7.2. However, dataobtained after administration of pharmaceutical formulations showed

[H 3 O+

]

N+

NN

+

CH3

N-

O

H H

N+

NN

+

CH3

N

-O

H H

protonated forms of nevirapine in aqueous medium.

-12

-10

-8

-6

-4

-2

0

2

4

0 50 100 150 200 250 300 350

Hea

t F

low

(W

/g)

Temperature (ºC)

Nevirapine

Nevirapine+Lactose

Nevirapine+Methocel K4M

Nevirapine+Kollidon VA64

Nevirapine+Kollidon K30

Nevirapine+Cellulose MC

Fig. 7. DSC curves of pure nevirapine and in physical mixture with the excipients (1:1).The curves were performed under inert atmosphere (N2) 50 mL/min, m=3.0 mg andβ=10 °C/min.

600 G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602

low release, with no significant differences in the bioavailability of thedrug. Thus, a dissolution test employing a surfactant concentration sim-ilar to that found under normal conditions in the TGAmay be oneway toevaluate and obtain results that are more representative of the likely be-haviour of the drug in vivo.

To study the impact of the presence of surfactants on the solubilityof nevirapine, assays were performed using phosphate buffer solu-tions with sodium lauryl sulphate added at concentrations of 0.1,0.5 and 1.0% (w/w). This methodology was employed to reduce thetime and cost requirements of the data acquisition and quantificationmethod. However, the presence of other constituents in the mediumthat mimic the gastrointestinal tract (e.g. lecithin and bile salts) wouldbe expected to favour the solubilization and subsequent absorption ofthe drug. One negative aspect of the employment of surfactants istheir impact on the method of quantification, especially because theymight interfere with the UV analysis. In the solubility tests, the quanti-fication of the samples containing the drug in the presence of surfactantwas carried out using the same spectrophotometric method as before,but a white solution containing surfactant was employed.

The data obtained from the solubility assay in the presence of so-dium lauryl sulphate (Fig. 6) showed that even at high pH (>4.5),under which conditions the drug has low molecular dissolution, thepresence of an additional compound to reduce the surface tensionand increase interaction with hydrophobic regions of the moleculefavoured the diffusion of nevirapine in the liquid phase. Using onlythe solubility data for pH=6, in which the pH is a critical factor, wewould expect that dissolution in the normal gastrointestinal tractwould most likely occur when the drug was precipitated by alkalini-zation of the stomach contents at the entrance to the duodenum,and this is a factor critical for absorption. However, even the highestconcentration (1%) of anionic surfactant used in the solubility mea-surements still does not generate surface tension values similar tothose produced by bile salts and cholesterol in vivo. Thus, it is likelythat the solubility of the drug is greater in the gastrointestinal tract[34].

The speed at which a solid dissolves in a dissolution medium or abiological fluid is a function of its surface area, solubility and dissolu-tion rate constant, and of the solute concentration in the medium. Be-cause of this, it is appropriate to study the behaviour of a drug duringthe dissolution process by carrying out the experiment in such a waythat the surface area is kept constant. This can be done by employingan apparatus for measuring the intrinsic dissolution rate. The IDR ofnevirapine was 0.4109 mg/cm2 min, according to a linear regressionof the data obtained in the test (y=0.4109x+0.4059; R2=1). Thedata obtained from the determination of the IDR can provide informa-tion about the behaviour of the drug prior to absorption, since disso-lution is a continuous phenomenon, with differences in solubility, andthese data can be better correlated with the dynamics of dissolutionin the gastrointestinal tract.

0

1

2

3

4

5

6

HCl 0.1 N HCl 0.01 N Buffer pH 6 Buffer pH 6 +0.1%

Buffer pH 6 +0.5%

Buffer pH 6 +1.0%

Solu

bilit

y (g

/L)

Fig. 6. Solubility of nevirapine (g/L) in various buffer solutions, and in buffer solutionsadded with 0.1, 0.5 and 1.0% sodium lauryl sulphate.

A drug may be considered highly soluble if it has an IDR above1.0 mg/cm2 min; in this case, bioavailability problems are less com-mon [35]. However, values of the IDR below 0.1 mg/cm2 min indicatethat bioavailability problems may arise because of drug-dependentdissolution.

When the data obtained from the IDR tests are compared with thedata from the measurements of the solubility in acid (0.1 N HCl), wecan see that the dissolution test was carried out under sink condi-tions, i.e. the maximum concentration of the drug was lower than15% of the solubility in the dissolution medium. Thus, the low intrin-sic dissolution value found was not influenced by saturation of themedium. The IDR varies fromdrug to drug andmay be related to the dif-fusion coefficient and the thickness of the film formed between thesolid surface and the liquid medium. If different batches of the samedrug are tested under the same conditions, the value of the intrinsic dis-solution constant will be affected only by factors inherent to the drug,such as themethod of synthesis, crystallinity, particle size and polymor-phism [36].

To study possible physical interactions and collect data to provideevidence of any incompatibility, nevirapine was evaluated by DSC andTGA both in the form of the pure drug and in the form of a 1:1 phys-ical mixture of the drug with an excipient. The excipients used wereKollidon® VA64, Kollidon® K30, hydroxypropylmethylcellulose, mi-crocrystalline cellulose PH 101 and lactose.

The thermoanalytical curve obtained from DSC shows that the drughas only one thermal event, which starts at around 240 °C, with tonset=244.48 °C and a peak at 246.79 °C extending up to 260 °C; this is relatedto the melting process, with an enthalpy determined as ΔH=130 J/g.After melting of the drug, no other event was observed until 300 °C

-30

-10

10

30

50

70

90

110

0 200 400 600 800

Wei

ght

(%)

Temperature (ºC)

NevirapineNevirapine+LactoseNevirapine+Methocel k4MNevirapine+Kollidon VA64Nevirapine+Kollidon K30Nevirapine+Celulose MC

Fig. 8. Thermogravimetric curves of nevirapine and physical mixtures in 1:1 (w/w) ofexcipients used in the compatibility test. Analyses were performed under inert atmo-sphere (N2) 50 mL/min, m=10.0±0.1 mg and β=10 °C/min.

Table 2Thermodynamic data obtained from the thermoanalytical curves DSC, TG and DTG ofmixture between nevirapine and excipients.

Nevirapine/excipients tmelting (°C) tonset TG 1° tpeak DTG 2° tpeak DTG

Nevirapine 246.48 286.03 250.37 329.56Nevirapine+cellulose MC 246.54 258.48 249.13 300.48Nevirapine+lactose 239.44 257.92 243.09 314.09Nevirapine+PVP K30 237.17 281.11 247.56 a

Nevirapine+PVP VA 64 233.52 282.07 254.94 305.33Nevirapine+Methocel K4M 242.63 282.07 248.16 318.89

a Non-observed peak due to overlapping.

601G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602

was reached. The thermoanalytical curve was also used to estimate thepurity of the drug, using the cryoscopic equation provided by the TAUniversal Analysis programme. The purity was found to be 99.0% orgreater; however, the correction had a high value (20%). The adequacyof the method for estimating the purity is highly dependent on the cor-rection,which should be below10% to provide reliable data. The use of alower weight and lower heating rate may be the reason for the 20%value. DSC curves of the mixtures of nevirapine with the various excip-ients are shown in Fig. 7. In general, when amixture of an excipient anda drug is employed, interaction may occur, and the physical propertiesof themixture and the dosage can be seriously affected. If there is no in-teraction, either physically or chemically, then themixture should showthe same thermal properties as the individual products [37].

The TGA profile was also obtained (Fig. 8). The thermogravimetriccurve indicates that the total mass loss occurs in only one event, whichstarts from about 200 °C with tonset=271.41 °C and extends up to320 °C. Amathematical treatment of the data using differentiation indi-cates that degradation of the drug occurs in at least two distinct steps.The first peak has tonset=238.47 °C and a residual mass of 94.59%; thepeak is at 246.98 °C, extending to 256.88 °C with a residual mass of87.63%. The second peak has tonset=306.30 °C and a residual mass of20.87%; the peak is at 315.69 °C, extending to 323.74 °C with 0.02% re-sidual mass. The analyses were performed under an inert atmosphere(N2) with a flow rate of 50 mL/min, 10.0±0.1 mg of mass (m) andcooling rate of 10 °C/min (β).

Thermodynamic data for nevirapine obtained from the DSC, TGAand differential thermogravimetry (DTG) thermoanalytical curvesare presented in Table 2.

When we observe the thermoanalytical profiles of pure nevirapineand of the binary mixtures with the excipients, the endothermic melt-ing event characteristic of the drug is present in all of the DSC curves.Themicrocrystalline cellulose and lactosemixtures show the character-istic melting peak of nevirapine, but with a clear shift relative to theinitial run. The mixtures with Kollidon® VA64, Kollidon® K30 andMethocel® K4M do not show the characteristic melting peak of nevira-pine; there is only a minor thermal event, shifted to the left and with areduced enthalpy. It is clear that all of the excipients, with the exceptionof microcrystalline cellulose, interact with the drug by changing itsthermoanalytical profile. The DSC analysis of lactose usually showsthree endothermic events due to the two most common forms oflactose, at 140, 220 and 240 °C, corresponding to the dehydration ofα-lactose and the melting of α- and β-lactose, respectively. When the

Table 3Data obtained after quantification of the drug nevirapine in different buffer solutions using speThe data presented were obtained in control through the use of solutions filled into ampoules

Solution Control Readings

HCl 0.1 N 0.1945 0.1941 0.1932 0.1HCl 0.01 N 0.2188 0.2172 0.2197 0.2Buffer 0.1 pH 4.5 0.2071 0.2064 0.2070 0.2Buffer 0.1 pH 7.2 0.2070 0.2055 0.2068 0.2

C=concentration control solution (μg/mL). A=concentration sample (μg/mL).

mixture is subjected to heating (Fig. 7), only one event is observed inthe temperature range 225–250 °C. Apparently, the melting of lactosecauses a displacement of the melting peak, promoting drug dissolutionand partial melting at a lower temperature. In general, this is not indic-ative of any potential incompatibility. Nevertheless, TGA was carriedout to provide data on the chemical and structural changes in order toclearly characterize this event (Fig. 8). The resulting thermograms ofthe mixtures are sums of the events related to the two components, in-cluding the degradation reaction and the mass loss involved. The dataobtained from the TGA curves have been summarized above.

The parameters used for the comparison of the thermoanalyticalcurves were the melting temperature, the onset temperature of weightloss (tonset TGA) and the temperature of maximum rate of mass loss(a typical TGA 1 and 2 event). The decrease in tonset TGA was attrib-uted to the presence of lactose in the binary mixture. The TGA peakwas also shifted to lower values. The Kollidon® 64 VA, Kollidon®K30 and Methocel® K4M mixtures also showed thermal events;however, the tonset TGA values were not significantly affected, indi-cating only physical interaction between these excipients and thedrug. This phenomenon was also reported by Grangeiro et al. [38]when PVP was used; it was explained by the formation of crystallinemicroaggregates of the drug and dispersion in a matrix of this amor-phous polymer.

The evaluation of a drug for degradation under light exposure shouldemploy tests that allow degradation products and/or structural changesof the drugmolecule to be identified [39]. Samples (pure nevirapine and1:1 physical mixtures with the excipients) were exposed to light beforefurther DSC and TGA analysis (Figs. 7 and 8). The powders were evalu-ated for changes in appearance in comparison with the product storedunder suitable storage conditions. After the exposure period, therewere no changes in the colour of either the drug powder or solutionsof it, where precipitation was also not observed. The data obtainedfrom DSC analysis showed no changes in the event related to the melt-ing of the drug (at 240 and 250 °C). The peaks related to this eventshowed similar endothermic peaks (at 246.58 and 246.23 °C) for sam-ples of nevirapine before and after exposure to light. The TGA data con-firmed the absence of changes. The overlapping curves indicated thatthere was no impact on the events related to mass loss or on thepeaks related to the derivative, indicating that neither the temperatureat which degradation occurs nor the rate at which degradation occurshad changed.

The samples were subjected to further quantification to assess theimpact of light on the samples in solution. The results are shown inTable 3. The data showed that light exposure did not cause severedegradation of the drug in aqueous media. However, in acid solutions,a decrease in the drug concentration was observed compared withthe samples in phosphate buffer media at two different pH values.After light exposure, the nevirapine was analysed further by DSCand TGA (Figs. 9 and 10).

4. Conclusions

Physicochemical analysis has been performed to obtain informa-tion about nevirapine prior to formulation. The results show that

ctrophotometric method. Readings were taken at λ=294 nm and 313 nm (0.01 N HCl).with similar feature used in the other samples but covered with aluminium.

Average Concentration (μg/mL) % of recovery

C A

937 0.1937 18.83 18.75 99.59179 0.2183 19.37 19.32 99.76077 0.2070 19.98 19.98 99.97071 0.2065 19.97 19.92 99.76

-6

-5

-3

-2

0

2

3

0 100 200 300 400

Hea

t F

low

(W

/g)

Temperature (ºC)

Nevirapine

Nevirapine photostability

Fig. 9. DSC of nevirapine before and after light exposure. The curve was performedunder inert atmosphere (N2) 50 mL/min, m≈2.0 mg and β=10 °C/min.

-0.40

-0.30

-0.20

-0.10

0.00

0.10

-20

0

20

40

60

80

100

120

140

0 100 200 300 400 500 600

2nd

Der

iv. w

eigh

t (%

/ºC

^2)

Wei

ght

(%)

Temperature (ºC)

Nevirapine

Nevirapine TGA

Nevirapine photo

Nevirapine photostabilityTGA

Fig. 10. First and second derived TGA of nevirapine before and after light exposure.Analyses were performed under inert atmosphere (N2) 50 mL/min, m=10.0±0.1 mg and β=10 °C/min.

602 G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602

the molecule has high stability in the solid state and is not susceptibleto degradation under light exposure. It has a high melting point(246 °C), which is the only event in the DSC profile, and low hygro-scopicity, facilitating handling of the drug in the normal environ-ment. An investigation of its interaction with other excipients (lactose,microcrystalline cellulose, PVP/PVA and HPMC) selected for their wideuse in solid dosage forms showed no incompatibilities. The drug haslow solubility, an acid medium being the most appropriate medium forassessing the release profile of solid dosage forms. From our tests, disso-lutionwas shown to be the critical factor affecting the bioavailability; thisis typical of a class II drug according to the BCS.

Acknowledgements

The authors wish to acknowledge support from the Fundação paraa Ciência e Tecnologia do Ministério da Ciência e Tecnologia underreference number ERA-Eula/0002/2009. Ms Severino received sponsor-ship from CAPES (Coordenação Aperfeiçoamento de Pessoal de Nivel

Superior) and FAPESP (Fundação de Amparo a Pesquisa do Estado deSão Paulo).

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