Materials Science and Engineering C 33 (2013) 596–602
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Compatibility studies of nevirapine in physical mixtures with excipients for oral 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, Brazil Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas, Campinas 13083-970, Brazil Department of Pharmaceutical Technology, Faculty of Health Sciences, Fernando Pessoa University, Porto 4200-150, Portugal d 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 b c
a r t i c l e
i n f o
Article history: Received 5 August 2011 Received in revised form 30 August 2012 Accepted 30 September 2012 Available online 6 October 2012 Keywords: Nevirapine Differential scanning calorimetry (DSC) Thermogravimetric analysis (TGA) Solubility Intrinsic dissolution Photostability
a b s t r a c t Nevirapine is a hydrophobic non-nucleoside reverse transcriptase inhibitor, used in first line regimens of highly active antiretroviral therapy (HAART). The drug has more than one crystalline form, which may have implications for its behaviour during production and also for its in vivo performance. This study was aimed at exploring the suitability of thermoanalytical methods for the solid-state characterization of commercial crystalline forms of nevirapine. The drug powder was characterized by ultraviolet spectrophotometry, stereoscopy, scanning electron microscopy, wide-angle X-ray diffraction, measurements of density, flowability, solubility and intrinsic dissolution rate (IDR), differential scanning calorimetry, thermogravimetric analysis, and photostability measurements. The results showed that nevirapine has high stability and is not susceptible to degradation under light exposure. The drug showed compatibility with the excipients tested (lactose, microcrystalline cellulose, polyvinylpyrrolidone and polyvinyl acetate copolymer (PVP/PVA), and hydroxypropylmethylcellulose (HPMC)). Nevirapine has low solubility, an acid medium being the most appropriate medium for assessing the release of the drug from dosage forms. However, the data obtained from IDR testing indicate that dissolution is the critical factor for the bioavailability of this drug. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The development of solid dosage forms for the purpose of oral administration of antiretroviral drugs to treat HIV infection by highly active antiretroviral therapy (HAART) is becoming increasingly common, since the HIV epidemic is still infecting a large number of individuals annually. Knowledge about novel pharmaceutical dosage forms is of paramount importance in this context [1–3]. Nevirapine is a hydrophobic nonnucleoside reverse transcriptase inhibitor, used in first line regimens of antiretroviral therapy [4,5]. The drug exists in more than one crystalline form, namely the hemiethanolate (form I), hemiacetonitrilate (form II), hemichloroformate (form III), hemi-THF solvate (form IV), mixed hemiethanolate hemihydrate (form V) and hemitoluenate (form VI), reflecting physicochemical changes during production, and this also affects the in vivo performance upon administration [6]. Because of the special properties of nevirapine, pre-formulation studies are required to test the compatibility between the drug and
⁎ Corresponding author at: Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637. E-mail address: [email protected] (E.B. Souto). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.030
excipients. Incompatibilities may lead to an accelerated loss of power, complexation, acid–base interaction or the formation of a eutectic mixture, resulting in low bioavailability and lack of stability [7,8]. In vitro dissolution testing is the most important method in the development of solid dosage forms [9–13]. It can be used to predict the performance of a dosage form, especially when drug release is the limiting factor in the absorption process [12,14]. The Biopharmaceutical Classification System (BCS) [15,16] is a tool that can be used to design a dissolution testing programme for predicting the dissolution rate, solubility and intestinal permeability. These parameters affect the in vivo performance of the drug, and therefore the pharmacological activity [17]. Examples of drugs that have been tested in this way, such as ayurvedic medicinal herbs [18], nateglinide [19], ketoprofen [20], benznidazole [9] and lercanidipine [21], have been reported in the literature. The aim of the present study was to evaluate the physicochemical characteristics of nevirapine relevant to further processing into solid dosage forms. The physicochemical properties of the drug powder were characterized by ultraviolet (UV) spectrophotometry, stereoscopy, scanning 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 dissolution 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). The polyvinylpyrrolidone copolymer/polyvinyl acetate (PVP/PVA) derivatives Kollidon® VA64 (vinylpyrrolidone–vinyl acetate copolymer, (C6H9NO)n × (C4H6O2)m) and Kollidon® K30 (ethenyl-2pyrrolidinone 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 Blanver Farmoquímica (Taboão da Serra, Brazil) and lactose was purchased from Pharmatose (São Paulo, Brazil). 2.2. Methods 2.2.1. Quantitative analysis of nevirapine The nevirapine was quantified by UV spectrophotometry (DU-640, Beckman Coulter, Fullerton, CA, USA); it was analysed at 294 nm against a calibration curve (R2 = 0.999), employing solutions of secondary standards in various buffers with pH values ranging from 1.2 to 7.0. 2.2.2. Stereoscopy and scanning electron microscopy The appearance of the solid bulk powder of the drug was analysed in a stereoscope (Motic SMZ® 168 T, coupled to a Fujifilm Finepix S5100® digital camera) at a magnification of 10–120×. The data obtained related to the colour and appearance of the solid material and the particle shape. Micrographs of nevirapine samples were obtained using a scanning electron microscope (LEO, model LEO440i, New York, USA). The surfaces of the samples were metallized with a gold coating using a sputter coater (Polaron, model SC 7620, Schwalbach, Germany) prior to recording the images. 2.2.3. Wide-angle X-ray diffraction WAXS analysis (Rigaku Ultima X-ray diffractometer, The Woodlands, TX, USA) was performed using monochromatic 1.5418 Å radiation from a tube with a copper target operated at 40 kV and 30 mA (1.2 kW), and a scintillation detector. The samples were fixed with gel-type amorphous HIVAC-G Shin-Etsu silicone. The pitch angle for each sample was 0.05°; the irradiation time was 5 s, with rotation at 30 rpm. The interlayer spacings d were calculated from the reflections using Bragg's equation as follows: d¼
λ sin2θ
where λ is the wavelength of the incident X-ray beam and θ is the scattering angle. The parameter d is the separation between the planes belonging to a particular set of planes in the crystal lattice structure. 2.2.4. Determination of density Accurately weighed 10 g amounts of nevirapine were sieved (mesh 20), and then transferred to beakers for analysis on a standard tap density tester (TAP-2, Logan Instruments Corporation, Somerset, NJ, USA). The parameters used for the tap density test were 2000 taps over a period of 10 min, which was found to be the minimum required to stabilize the final volume. The initial volume (Vi) was recorded, followed by measurements after 1, 2, 5 and 10 min. These measurements were used to calculate the final volume (Vf). The apparent density (dap) and compacted density (dcp) were then calculated from the following equations: dap ¼ M i =V i dcp ¼ Mi =V f
597
where dap is in g/cm 3, Vi is in cm 3, Mi is the initial mass (in g), dcp is in g/cm 3 and Vf is in cm 3. Determination of the true density was performed using a helium pycnometer (Quantacrome model 1000, Boynton Beach, FL, USA). Accurately weighted 1.0 g samples of nevirapine were analysed using a small crucible, after a dehumidifying flow of helium had been applied for 3 min. Five readings were taken, with a variation of less than 0.01% standard error. The true density (ρ) was calculated as the ratio of the mass (m) to the volume (V), using the following equation: ρ¼
m : V
2.2.5. Flowability To assess the flowability of mixtures of nevirapine and excipients as used in pellets, the compressibility index (CpI), the Carr index and the Hausner ratio were determined as follows: CpI ¼
dcp −dap dcp
Carr index ¼
!
dcp −dap dcp
Hausner ratio ¼
! 100
dcp dap
where dap is the apparent density (g/cm 3) and dcp is the compacted density (g/cm 3). 2.2.6. Solubility To determine the maximum solubility of nevirapine, an excess of the drug (~250 mg) was transferred to vials containing a series of aqueous solutions of hydrochloric acid (from 0.1 to 0.01 M) and 0.1 M phosphate buffer solutions (at varying pH from 1.2 to 7.2) with stirring at 200 rpm for 72 h, at a constant temperature of 37 °C in a shaker (Tecnal, model TE-420, Piracicaba, Brazil). The samples were then centrifuged and quantified by spectrophotometry at 294 nm. The experiments were carried out in triplicate. 2.2.7. Effect of surfactant on solubility To study the impact of surfactants on the solubility of nevirapine, tests were carried out in phosphate buffers in the presence of the anionic surfactant sodium lauryl sulphate at concentrations of 0.1, 0.5 and 1.0% (w/w). This procedure was used because there was interference between the absorption of the excipients and the absorption of the drug at a wavelength of 294 nm. 2.2.8. Intrinsic dissolution rate To determine the intrinsic dissolution rate, a device designed by us was coupled to a conventional dissolution test apparatus. Samples of nevirapine (200 mg) were transferred to the test apparatus by subjecting them to compression (1000 kg/cm2) in a hydraulic press (equipped with 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 a temperature of 37 °C. The experiments were performed in triplicate for each condition, using a modified Wood apparatus (Varian Inc., Vankel Division, Cary, NC, USA). 2.2.9. Differential scanning calorimetry Characterization of nevirapine and of physical mixtures of nevirapine and excipients was performed by thermal analysis in a TA-2920 apparatus (TA Instruments, New Castle, DE, USA) using a temperature
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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×.
range from 25 to 300 °C. A sample mass of about 3.0 mg was weighed in a tightly closed aluminium container, and then analysed at a heating rate of 10 °C/min under a dynamic atmosphere of nitrogen with a flow rate of 50 mL/min. The thermodynamic parameters were analysed using the Universal Analysis® programme from TA.
4000
2.2.10. Thermogravimetric analysis Characterization of nevirapine and of physical mixtures of nevirapine and excipients was performed by thermogravimetry using a thermoanalytical balance (TA-2950, TA Instruments, New Castle, DE, USA) over a temperature range from 25 to 600 °C. A platinum crucible containing a sample mass of 10.0 mg was used at a heating rate of 10 °C/min under a dynamic nitrogen atmosphere with a flow rate of 100 mL/min. The data were analysed using the Universal Analysis® programme.
Lin (cps)
3000 2000 1000 0 0
10
20
30
40
50
2 theta
Volume (cm3)
20
0.80
16
0.60
12 0.40 8 0.20
4 0 0
1
2
5
10
15
Compact desnsity (g/cm3)
Fig. 2. WAXD diffractogram of neviparine.
0.00
Time (min) Fig. 3. Volume (columns) and compact density (line) versus time analysed during 15 min.
2.2.11. Photostability The 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 standard 20 μg/mL solutions in several different buffers, was subjected to light exposure for 24 h. This period was chosen after calibration of the equipment according to the actinometrical method. This method is based on the quantification of a solution of 2% (w/v) quinine monohydrochloride before exposure (at t = 0) and at predetermined time intervals until 0.9 absorbance is reached. The degradation of the nevirapine was checked following UV spectrophotometry, DSC and TGA. Table 1 Data obtained with the helium pycnometer equipment related to the different readings of a sample of nevirapine (m = 0.2207 g). Readings
Volume
True density
1 2 3 4 5 Average True density
0.1174 0.1362 0.1222 0.1376 0.1357 0.1365 cm3
1.8806 1.8059 1.6201 1.6041 1.6268 1.6170 g/cm3 ± 0.0008%
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8
6
7 6
4
5 4
3
3
2
Buffer pH
Solubility (g/L)
5
2 1 0
1 0 HCl 0.1 N
HCl 0.01 N Buffer pH 4.5 Buffer pH 6.0 Buffer pH 6.8
water
Buffer pH 7.2
Fig. 4. Values of solubility of nevirapine (g/L) in various buffer solutions. The pH values of the media in which the tests were conducted are also presented.
3. Results and discussion Using stereoscopy to analyse the bulk powder, the nevirapine appeared to be a partially crystalline material (Fig. 1). When the magnification was increased, more detail was seen and elongated particles were observed. Using SEM analysis, further details of a continuous mass of powder were obtained, consistent with a crystalline structure. Similar results were obtained by Chadha et al. [6] and Mohammed et al. [25] in analyses of commercial nevirapine, where the crystalline form of the drug was not clearly defined. To confirm the crystallinity of the nevirapine powder, WAXD analysis was carried out (Fig. 2). Interlayer spacings d of 9.2695, 13.3381 and 25.2979 Å were observed. These results characterize form I of nevirapine [26]. Flowability is an important factor in several processes in the pharmaceutical industry, including the compression process. Fig. 3 shows the results for the apparent and compacted density. A reduction in the volume of the powder was observed soon after the start of the test, and the volume stabilized after 5 min. The use of 2000 taps is usually recommended for measurements of the compacted density; this number was obtained by checking for changes in volume during tests until there was stabilization of the observed volume [27]. The result for the density of the compacted powder in our tests was 0.5748 g/cm3. The compressibility index CpI is related to the accommodation of the particles [28]. The result for the Hausner ratio was 1.45, and that for the Carr index was 31.24%. A Hausner ratio below 1.25 indicates good compressibility. The Carr index may be indicative of the flowability and degree of packing of a material, which are relevant properties when the matrices of a tableting machine are being filled. A Carr index lower than 15% indicates an adequate flow of the powder and stable packing, whereas values above 25% are characteristic of poor flow properties [29].
599
To improve flowability, changes in the crystal structure or in the particle size have been suggested, these being criteria for comparison between different batches of raw material. The data obtained with the helium pycnometer are shown in Table 1. The volume and true density obtained for the nevirapine were 0.1365 cm 3 ± 0.009329 and 1.6170 g/cm 3 ± 0.0008%, respectively. There is a significant difference between this value for the density and the data presented earlier for the compacted density of the same material. The difference between the two data sets is related to how they were obtained. The compacted density takes account of the total volume occupied by the powder. Although the particles are in contact, however, empty spaces still exist between the particles even after the compaction operation. The data from the pycnometer are obtained from the difference between the volumes occupied by the inert gas (He) in the crucible container with and without the powder. The empty space in the powder is occupied by the gas, and the true density of the drug is determined from the difference in volume. Solubility is an important parameter both for the in vitro evaluation of solid dosage forms and for proper dissolution and subsequent absorption in vivo. According to the BCS, for a drug to be considered highly soluble, a dose/solubility value of less than 250 mL is necessary over the whole pH range to which the drug will be exposed in the gastrointestinal tract. Data obtained, after equilibration, for the solubility of the drug in various buffer media are shown in Fig. 4. According to the solubility data and based on the usual dose of the drug (200 mg), it can be estimated that only the conditions present in the stomach (pH b 2) will be sufficient for the complete dissolution of nevirapine. The dose/solubility value in other media is greater than 250 mL. According to the BCS, this value defines the drug as poorly soluble, and, possibly, the step of dissolution of the drug from the dosage form may interfere with its bioavailability [30,31]. From Fig. 5, it can be seen that nevirapine is sensitive to pH, and this is one of the factors contributing to its dissolution. In an acid medium containing >2.10 g/L [H3O+], the solubility of the drug was 4.99 g/L. When the environment was changed using solutions with different pH values, the solubility of the drug was reduced by a factor of 10–40. Structural changes in the drug molecule in solution are responsible for the different solubility values at different pH values. The electron donor sites (nucleophiles) in nevirapine structure, represented by the pyridine nitrogen atoms, can be protonated under acidic conditions, forming a stable zwitterion form. By reducing the concentration of H3O+ ions, this form tends to stabilize the molecule [32]. Aungst et al. [33] studied an antiretroviral drug (DPC 961) of low solubility and high permeability in vitro; the dissolution was clearly impaired in media with pH values from 4.5 to 7.2. However, data obtained after administration of pharmaceutical formulations showed
H
H N
+
N
N
+
NN
N
N
+
[H 3 O ]
+ N H O
CH 3
CH 3
O
H2O
H
H N
+
N
N
+
N -O
CH 3
Fig. 5. Diagram representing the balance of molecular and protonated forms of nevirapine in aqueous medium.
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low release, with no significant differences in the bioavailability of the drug. Thus, a dissolution test employing a surfactant concentration similar to that found under normal conditions in the TGA may be one way to evaluate and obtain results that are more representative of the likely behaviour of the drug in vivo. To study the impact of the presence of surfactants on the solubility of nevirapine, assays were performed using phosphate buffer solutions with sodium lauryl sulphate added at concentrations of 0.1, 0.5 and 1.0% (w/w). This methodology was employed to reduce the time and cost requirements of the data acquisition and quantification method. However, the presence of other constituents in the medium that mimic the gastrointestinal tract (e.g. lecithin and bile salts) would be expected to favour the solubilization and subsequent absorption of the drug. One negative aspect of the employment of surfactants is their impact on the method of quantification, especially because they might interfere with the UV analysis. In the solubility tests, the quantification of the samples containing the drug in the presence of surfactant was 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 sodium lauryl sulphate (Fig. 6) showed that even at high pH (> 4.5), under which conditions the drug has low molecular dissolution, the presence of an additional compound to reduce the surface tension and increase interaction with hydrophobic regions of the molecule favoured the diffusion of nevirapine in the liquid phase. Using only the solubility data for pH = 6, in which the pH is a critical factor, we would expect that dissolution in the normal gastrointestinal tract would most likely occur when the drug was precipitated by alkalinization of the stomach contents at the entrance to the duodenum, and this is a factor critical for absorption. However, even the highest concentration (1%) of anionic surfactant used in the solubility measurements still does not generate surface tension values similar to those produced by bile salts and cholesterol in vivo. Thus, it is likely that the solubility of the drug is greater in the gastrointestinal tract [34]. The speed at which a solid dissolves in a dissolution medium or a biological fluid is a function of its surface area, solubility and dissolution rate constant, and of the solute concentration in the medium. Because of this, it is appropriate to study the behaviour of a drug during the dissolution process by carrying out the experiment in such a way that the surface area is kept constant. This can be done by employing an apparatus for measuring the intrinsic dissolution rate. The IDR of nevirapine was 0.4109 mg/cm 2 min, according to a linear regression of the data obtained in the test (y = 0.4109x + 0.4059; R 2 = 1). The data obtained from the determination of the IDR can provide information about the behaviour of the drug prior to absorption, since dissolution is a continuous phenomenon, with differences in solubility, and these data can be better correlated with the dynamics of dissolution in the gastrointestinal tract.
4
Nevirapine
2
Heat Flow (W/g)
600
Nevirapine+Lactose
0 -2
Nevirapine+Methocel K4M
-4
Nevirapine+Kollidon VA64
-6
Nevirapine+Kollidon K30
-8
Nevirapine+Cellulose MC
-10 -12 0
50
100
150
200
250
300
350
Temperature (ºC) 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 and β = 10 °C/min.
A drug may be considered highly soluble if it has an IDR above 1.0 mg/cm 2 min; in this case, bioavailability problems are less common [35]. However, values of the IDR below 0.1 mg/cm 2 min indicate that bioavailability problems may arise because of drug-dependent dissolution. When the data obtained from the IDR tests are compared with the data from the measurements of the solubility in acid (0.1 N HCl), we can see that the dissolution test was carried out under sink conditions, i.e. the maximum concentration of the drug was lower than 15% of the solubility in the dissolution medium. Thus, the low intrinsic dissolution value found was not influenced by saturation of the medium. The IDR varies from drug to drug and may be related to the diffusion coefficient and the thickness of the film formed between the solid surface and the liquid medium. If different batches of the same drug are tested under the same conditions, the value of the intrinsic dissolution constant will be affected only by factors inherent to the drug, such as the method of synthesis, crystallinity, particle size and polymorphism [36]. To study possible physical interactions and collect data to provide evidence of any incompatibility, nevirapine was evaluated by DSC and TGA both in the form of the pure drug and in the form of a 1:1 physical mixture of the drug with an excipient. The excipients used were Kollidon® VA64, Kollidon® K30, hydroxypropylmethylcellulose, microcrystalline cellulose PH 101 and lactose. The thermoanalytical curve obtained from DSC shows that the drug has 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 related to 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 110
6
Weight (%)
5
Solubility (g/L)
Nevirapine Nevirapine+Lactose Nevirapine+Methocel k4M Nevirapine+Kollidon VA64 Nevirapine+Kollidon K30 Nevirapine+Celulose MC
90
4 3
70 50 30 10
2 -10 1
-30 0
0 HCl 0.1 N
HCl 0.01 N
Buffer pH 6
Buffer pH 6 + Buffer pH 6 + Buffer pH 6 + 0.1% 0.5% 1.0%
Fig. 6. Solubility of nevirapine (g/L) in various buffer solutions, and in buffer solutions added with 0.1, 0.5 and 1.0% sodium lauryl sulphate.
200
400
600
800
Temperature (ºC) Fig. 8. Thermogravimetric curves of nevirapine and physical mixtures in 1:1 (w/w) of excipients used in the compatibility test. Analyses were performed under inert atmosphere (N2) 50 mL/min, m= 10.0 ± 0.1 mg and β = 10 °C/min.
G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602 Table 2 Thermodynamic data obtained from the thermoanalytical curves DSC, TG and DTG of mixture between nevirapine and excipients. Nevirapine/excipients
tmelting (°C)
tonset TG
1° tpeak DTG
2° tpeak DTG
Nevirapine Nevirapine + cellulose MC Nevirapine + lactose Nevirapine + PVP K30 Nevirapine + PVP VA 64 Nevirapine + Methocel K4M
246.48 246.54 239.44 237.17 233.52 242.63
286.03 258.48 257.92 281.11 282.07 282.07
250.37 249.13 243.09 247.56 254.94 248.16
329.56 300.48 314.09
a
a
305.33 318.89
Non-observed peak due to overlapping.
was reached. The thermoanalytical curve was also used to estimate the purity of the drug, using the cryoscopic equation provided by the TA Universal Analysis programme. The purity was found to be 99.0% or greater; however, the correction had a high value (20%). The adequacy of the method for estimating the purity is highly dependent on the correction, which should be below 10% to provide reliable data. The use of a lower weight and lower heating rate may be the reason for the 20% value. DSC curves of the mixtures of nevirapine with the various excipients are shown in Fig. 7. In general, when a mixture of an excipient and a drug is employed, interaction may occur, and the physical properties of the mixture and the dosage can be seriously affected. If there is no interaction, either physically or chemically, then the mixture should show the same thermal properties as the individual products [37]. The TGA profile was also obtained (Fig. 8). The thermogravimetric curve indicates that the total mass loss occurs in only one event, which starts from about 200 °C with tonset = 271.41 °C and extends up to 320 °C. A mathematical treatment of the data using differentiation indicates 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%; the peak is at 246.98 °C, extending to 256.88 °C with a residual mass of 87.63%. The second peak has tonset = 306.30 °C and a residual mass of 20.87%; the peak is at 315.69 °C, extending to 323.74 °C with 0.02% residual 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) and cooling rate of 10 °C/min (β). Thermodynamic data for nevirapine obtained from the DSC, TGA and differential thermogravimetry (DTG) thermoanalytical curves are presented in Table 2. When we observe the thermoanalytical profiles of pure nevirapine and of the binary mixtures with the excipients, the endothermic melting event characteristic of the drug is present in all of the DSC curves. The microcrystalline cellulose and lactose mixtures show the characteristic melting peak of nevirapine, but with a clear shift relative to the initial run. The mixtures with Kollidon® VA64, Kollidon® K30 and Methocel® K4M do not show the characteristic melting peak of nevirapine; there is only a minor thermal event, shifted to the left and with a reduced enthalpy. It is clear that all of the excipients, with the exception of microcrystalline cellulose, interact with the drug by changing its thermoanalytical profile. The DSC analysis of lactose usually shows three endothermic events due to the two most common forms of lactose, at 140, 220 and 240 °C, corresponding to the dehydration of α-lactose and the melting of α- and β-lactose, respectively. When the
601
mixture is subjected to heating (Fig. 7), only one event is observed in the temperature range 225–250 °C. Apparently, the melting of lactose causes a displacement of the melting peak, promoting drug dissolution and partial melting at a lower temperature. In general, this is not indicative of any potential incompatibility. Nevertheless, TGA was carried out to provide data on the chemical and structural changes in order to clearly characterize this event (Fig. 8). The resulting thermograms of the mixtures are sums of the events related to the two components, including the degradation reaction and the mass loss involved. The data obtained from the TGA curves have been summarized above. The parameters used for the comparison of the thermoanalytical curves were the melting temperature, the onset temperature of weight loss (tonset TGA) and the temperature of maximum rate of mass loss (a typical TGA 1 and 2 event). The decrease in tonset TGA was attributed to the presence of lactose in the binary mixture. The TGA peak was 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, indicating only physical interaction between these excipients and the drug. This phenomenon was also reported by Grangeiro et al. [38] when PVP was used; it was explained by the formation of crystalline microaggregates of the drug and dispersion in a matrix of this amorphous polymer. The evaluation of a drug for degradation under light exposure should employ tests that allow degradation products and/or structural changes of the drug molecule to be identified [39]. Samples (pure nevirapine and 1:1 physical mixtures with the excipients) were exposed to light before further DSC and TGA analysis (Figs. 7 and 8). The powders were evaluated for changes in appearance in comparison with the product stored under suitable storage conditions. After the exposure period, there were no changes in the colour of either the drug powder or solutions of it, where precipitation was also not observed. The data obtained from DSC analysis showed no changes in the event related to the melting of the drug (at 240 and 250 °C). The peaks related to this event showed similar endothermic peaks (at 246.58 and 246.23 °C) for samples of nevirapine before and after exposure to light. The TGA data confirmed the absence of changes. The overlapping curves indicated that there was no impact on the events related to mass loss or on the peaks related to the derivative, indicating that neither the temperature at which degradation occurs nor the rate at which degradation occurs had changed. The samples were subjected to further quantification to assess the impact of light on the samples in solution. The results are shown in Table 3. The data showed that light exposure did not cause severe degradation of the drug in aqueous media. However, in acid solutions, a decrease in the drug concentration was observed compared with the samples in phosphate buffer media at two different pH values. After light exposure, the nevirapine was analysed further by DSC and TGA (Figs. 9 and 10). 4. Conclusions Physicochemical analysis has been performed to obtain information about nevirapine prior to formulation. The results show that
Table 3 Data obtained after quantification of the drug nevirapine in different buffer solutions using spectrophotometric method. Readings were taken at λ=294 nm and 313 nm (0.01 N HCl). The data presented were obtained in control through the use of solutions filled into ampoules with similar feature used in the other samples but covered with aluminium. Solution
HCl 0.1 N HCl 0.01 N Buffer 0.1 pH 4.5 Buffer 0.1 pH 7.2
Control
0.1945 0.2188 0.2071 0.2070
Readings
0.1941 0.2172 0.2064 0.2055
Average
0.1932 0.2197 0.2070 0.2068
C = concentration control solution (μg/mL). A= concentration sample (μg/mL).
0.1937 0.2179 0.2077 0.2071
0.1937 0.2183 0.2070 0.2065
Concentration (μg/mL) C
A
18.83 19.37 19.98 19.97
18.75 19.32 19.98 19.92
% of recovery
99.59 99.76 99.97 99.76
602
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3
Superior) and FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo).
Nevirapine Heat Flow (W/g)
2
References
Nevirapine photostability 0 -2 -3 -5 -6 0
100
200
300
400
Temperature (ºC) Fig. 9. DSC of nevirapine before and after light exposure. The curve was performed under inert atmosphere (N2) 50 mL/min, m ≈ 2.0 mg and β = 10 °C/min.
0.10
140
0.00
Weight (%)
100 80
-0.10
60 Nevirapine
40
-0.20
Nevirapine TGA
20
Nevirapine photo
0
-0.30
2nd Deriv. weight (%/ºC^2)
120
Nevirapine photostability TGA
-20
-0.40 0
100
200 300 400 Temperature (ºC)
500
600
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.
the molecule has high stability in the solid state and is not susceptible to degradation under light exposure. It has a high melting point (246 °C), which is the only event in the DSC profile, and low hygroscopicity, facilitating handling of the drug in the normal environment. An investigation of its interaction with other excipients (lactose, microcrystalline cellulose, PVP/PVA and HPMC) selected for their wide use in solid dosage forms showed no incompatibilities. The drug has low solubility, an acid medium being the most appropriate medium for assessing the release profile of solid dosage forms. From our tests, dissolution was shown to be the critical factor affecting the bioavailability; this is typical of a class II drug according to the BCS.
Acknowledgements The authors wish to acknowledge support from the Fundação para a Ciência e Tecnologia do Ministério da Ciência e Tecnologia under reference number ERA-Eula/0002/2009. Ms Severino received sponsorship from CAPES (Coordenação Aperfeiçoamento de Pessoal de Nivel
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Compatibility studies of nevirapine in physical mixtures with excipients for oral 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, Brazil Department of Biotechnological Processes, School of Chemical Engineering, University of Campinas, Campinas 13083-970, Brazil Department of Pharmaceutical Technology, Faculty of Health Sciences, Fernando Pessoa University, Porto 4200-150, Portugal d 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 b c
a r t i c l e
i n f o
Article history: Received 5 August 2011 Received in revised form 30 August 2012 Accepted 30 September 2012 Available online 6 October 2012 Keywords: Nevirapine Differential scanning calorimetry (DSC) Thermogravimetric analysis (TGA) Solubility Intrinsic dissolution Photostability
a b s t r a c t Nevirapine is a hydrophobic non-nucleoside reverse transcriptase inhibitor, used in first line regimens of highly active antiretroviral therapy (HAART). The drug has more than one crystalline form, which may have implications for its behaviour during production and also for its in vivo performance. This study was aimed at exploring the suitability of thermoanalytical methods for the solid-state characterization of commercial crystalline forms of nevirapine. The drug powder was characterized by ultraviolet spectrophotometry, stereoscopy, scanning electron microscopy, wide-angle X-ray diffraction, measurements of density, flowability, solubility and intrinsic dissolution rate (IDR), differential scanning calorimetry, thermogravimetric analysis, and photostability measurements. The results showed that nevirapine has high stability and is not susceptible to degradation under light exposure. The drug showed compatibility with the excipients tested (lactose, microcrystalline cellulose, polyvinylpyrrolidone and polyvinyl acetate copolymer (PVP/PVA), and hydroxypropylmethylcellulose (HPMC)). Nevirapine has low solubility, an acid medium being the most appropriate medium for assessing the release of the drug from dosage forms. However, the data obtained from IDR testing indicate that dissolution is the critical factor for the bioavailability of this drug. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The development of solid dosage forms for the purpose of oral administration of antiretroviral drugs to treat HIV infection by highly active antiretroviral therapy (HAART) is becoming increasingly common, since the HIV epidemic is still infecting a large number of individuals annually. Knowledge about novel pharmaceutical dosage forms is of paramount importance in this context [1–3]. Nevirapine is a hydrophobic nonnucleoside reverse transcriptase inhibitor, used in first line regimens of antiretroviral therapy [4,5]. The drug exists in more than one crystalline form, namely the hemiethanolate (form I), hemiacetonitrilate (form II), hemichloroformate (form III), hemi-THF solvate (form IV), mixed hemiethanolate hemihydrate (form V) and hemitoluenate (form VI), reflecting physicochemical changes during production, and this also affects the in vivo performance upon administration [6]. Because of the special properties of nevirapine, pre-formulation studies are required to test the compatibility between the drug and
⁎ Corresponding author at: Faculty of Health Sciences, Fernando Pessoa University, Rua Carlos da Maia 296, Office S.1, P-4200-150 Porto, Portugal. Tel.: +351 22 507 4630x3056; fax: +351 22 550 4637. E-mail address: [email protected] (E.B. Souto). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2012.09.030
excipients. Incompatibilities may lead to an accelerated loss of power, complexation, acid–base interaction or the formation of a eutectic mixture, resulting in low bioavailability and lack of stability [7,8]. In vitro dissolution testing is the most important method in the development of solid dosage forms [9–13]. It can be used to predict the performance of a dosage form, especially when drug release is the limiting factor in the absorption process [12,14]. The Biopharmaceutical Classification System (BCS) [15,16] is a tool that can be used to design a dissolution testing programme for predicting the dissolution rate, solubility and intestinal permeability. These parameters affect the in vivo performance of the drug, and therefore the pharmacological activity [17]. Examples of drugs that have been tested in this way, such as ayurvedic medicinal herbs [18], nateglinide [19], ketoprofen [20], benznidazole [9] and lercanidipine [21], have been reported in the literature. The aim of the present study was to evaluate the physicochemical characteristics of nevirapine relevant to further processing into solid dosage forms. The physicochemical properties of the drug powder were characterized by ultraviolet (UV) spectrophotometry, stereoscopy, scanning 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 dissolution rate (IDR) were also carried out on the bulk material.
G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602
2. Material and methods 2.1. Material Nevirapine was provided by Cristália (Itapira, Brazil). The polyvinylpyrrolidone copolymer/polyvinyl acetate (PVP/PVA) derivatives Kollidon® VA64 (vinylpyrrolidone–vinyl acetate copolymer, (C6H9NO)n × (C4H6O2)m) and Kollidon® K30 (ethenyl-2pyrrolidinone 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 Blanver Farmoquímica (Taboão da Serra, Brazil) and lactose was purchased from Pharmatose (São Paulo, Brazil). 2.2. Methods 2.2.1. Quantitative analysis of nevirapine The nevirapine was quantified by UV spectrophotometry (DU-640, Beckman Coulter, Fullerton, CA, USA); it was analysed at 294 nm against a calibration curve (R2 = 0.999), employing solutions of secondary standards in various buffers with pH values ranging from 1.2 to 7.0. 2.2.2. Stereoscopy and scanning electron microscopy The appearance of the solid bulk powder of the drug was analysed in a stereoscope (Motic SMZ® 168 T, coupled to a Fujifilm Finepix S5100® digital camera) at a magnification of 10–120×. The data obtained related to the colour and appearance of the solid material and the particle shape. Micrographs of nevirapine samples were obtained using a scanning electron microscope (LEO, model LEO440i, New York, USA). The surfaces of the samples were metallized with a gold coating using a sputter coater (Polaron, model SC 7620, Schwalbach, Germany) prior to recording the images. 2.2.3. Wide-angle X-ray diffraction WAXS analysis (Rigaku Ultima X-ray diffractometer, The Woodlands, TX, USA) was performed using monochromatic 1.5418 Å radiation from a tube with a copper target operated at 40 kV and 30 mA (1.2 kW), and a scintillation detector. The samples were fixed with gel-type amorphous HIVAC-G Shin-Etsu silicone. The pitch angle for each sample was 0.05°; the irradiation time was 5 s, with rotation at 30 rpm. The interlayer spacings d were calculated from the reflections using Bragg's equation as follows: d¼
λ sin2θ
where λ is the wavelength of the incident X-ray beam and θ is the scattering angle. The parameter d is the separation between the planes belonging to a particular set of planes in the crystal lattice structure. 2.2.4. Determination of density Accurately weighed 10 g amounts of nevirapine were sieved (mesh 20), and then transferred to beakers for analysis on a standard tap density tester (TAP-2, Logan Instruments Corporation, Somerset, NJ, USA). The parameters used for the tap density test were 2000 taps over a period of 10 min, which was found to be the minimum required to stabilize the final volume. The initial volume (Vi) was recorded, followed by measurements after 1, 2, 5 and 10 min. These measurements were used to calculate the final volume (Vf). The apparent density (dap) and compacted density (dcp) were then calculated from the following equations: dap ¼ M i =V i dcp ¼ Mi =V f
597
where dap is in g/cm 3, Vi is in cm 3, Mi is the initial mass (in g), dcp is in g/cm 3 and Vf is in cm 3. Determination of the true density was performed using a helium pycnometer (Quantacrome model 1000, Boynton Beach, FL, USA). Accurately weighted 1.0 g samples of nevirapine were analysed using a small crucible, after a dehumidifying flow of helium had been applied for 3 min. Five readings were taken, with a variation of less than 0.01% standard error. The true density (ρ) was calculated as the ratio of the mass (m) to the volume (V), using the following equation: ρ¼
m : V
2.2.5. Flowability To assess the flowability of mixtures of nevirapine and excipients as used in pellets, the compressibility index (CpI), the Carr index and the Hausner ratio were determined as follows: CpI ¼
dcp −dap dcp
Carr index ¼
!
dcp −dap dcp
Hausner ratio ¼
! 100
dcp dap
where dap is the apparent density (g/cm 3) and dcp is the compacted density (g/cm 3). 2.2.6. Solubility To determine the maximum solubility of nevirapine, an excess of the drug (~250 mg) was transferred to vials containing a series of aqueous solutions of hydrochloric acid (from 0.1 to 0.01 M) and 0.1 M phosphate buffer solutions (at varying pH from 1.2 to 7.2) with stirring at 200 rpm for 72 h, at a constant temperature of 37 °C in a shaker (Tecnal, model TE-420, Piracicaba, Brazil). The samples were then centrifuged and quantified by spectrophotometry at 294 nm. The experiments were carried out in triplicate. 2.2.7. Effect of surfactant on solubility To study the impact of surfactants on the solubility of nevirapine, tests were carried out in phosphate buffers in the presence of the anionic surfactant sodium lauryl sulphate at concentrations of 0.1, 0.5 and 1.0% (w/w). This procedure was used because there was interference between the absorption of the excipients and the absorption of the drug at a wavelength of 294 nm. 2.2.8. Intrinsic dissolution rate To determine the intrinsic dissolution rate, a device designed by us was coupled to a conventional dissolution test apparatus. Samples of nevirapine (200 mg) were transferred to the test apparatus by subjecting them to compression (1000 kg/cm2) in a hydraulic press (equipped with 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 a temperature of 37 °C. The experiments were performed in triplicate for each condition, using a modified Wood apparatus (Varian Inc., Vankel Division, Cary, NC, USA). 2.2.9. Differential scanning calorimetry Characterization of nevirapine and of physical mixtures of nevirapine and excipients was performed by thermal analysis in a TA-2920 apparatus (TA Instruments, New Castle, DE, USA) using a temperature
598
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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×.
range from 25 to 300 °C. A sample mass of about 3.0 mg was weighed in a tightly closed aluminium container, and then analysed at a heating rate of 10 °C/min under a dynamic atmosphere of nitrogen with a flow rate of 50 mL/min. The thermodynamic parameters were analysed using the Universal Analysis® programme from TA.
4000
2.2.10. Thermogravimetric analysis Characterization of nevirapine and of physical mixtures of nevirapine and excipients was performed by thermogravimetry using a thermoanalytical balance (TA-2950, TA Instruments, New Castle, DE, USA) over a temperature range from 25 to 600 °C. A platinum crucible containing a sample mass of 10.0 mg was used at a heating rate of 10 °C/min under a dynamic nitrogen atmosphere with a flow rate of 100 mL/min. The data were analysed using the Universal Analysis® programme.
Lin (cps)
3000 2000 1000 0 0
10
20
30
40
50
2 theta
Volume (cm3)
20
0.80
16
0.60
12 0.40 8 0.20
4 0 0
1
2
5
10
15
Compact desnsity (g/cm3)
Fig. 2. WAXD diffractogram of neviparine.
0.00
Time (min) Fig. 3. Volume (columns) and compact density (line) versus time analysed during 15 min.
2.2.11. Photostability The 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 standard 20 μg/mL solutions in several different buffers, was subjected to light exposure for 24 h. This period was chosen after calibration of the equipment according to the actinometrical method. This method is based on the quantification of a solution of 2% (w/v) quinine monohydrochloride before exposure (at t = 0) and at predetermined time intervals until 0.9 absorbance is reached. The degradation of the nevirapine was checked following UV spectrophotometry, DSC and TGA. Table 1 Data obtained with the helium pycnometer equipment related to the different readings of a sample of nevirapine (m = 0.2207 g). Readings
Volume
True density
1 2 3 4 5 Average True density
0.1174 0.1362 0.1222 0.1376 0.1357 0.1365 cm3
1.8806 1.8059 1.6201 1.6041 1.6268 1.6170 g/cm3 ± 0.0008%
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8
6
7 6
4
5 4
3
3
2
Buffer pH
Solubility (g/L)
5
2 1 0
1 0 HCl 0.1 N
HCl 0.01 N Buffer pH 4.5 Buffer pH 6.0 Buffer pH 6.8
water
Buffer pH 7.2
Fig. 4. Values of solubility of nevirapine (g/L) in various buffer solutions. The pH values of the media in which the tests were conducted are also presented.
3. Results and discussion Using stereoscopy to analyse the bulk powder, the nevirapine appeared to be a partially crystalline material (Fig. 1). When the magnification was increased, more detail was seen and elongated particles were observed. Using SEM analysis, further details of a continuous mass of powder were obtained, consistent with a crystalline structure. Similar results were obtained by Chadha et al. [6] and Mohammed et al. [25] in analyses of commercial nevirapine, where the crystalline form of the drug was not clearly defined. To confirm the crystallinity of the nevirapine powder, WAXD analysis was carried out (Fig. 2). Interlayer spacings d of 9.2695, 13.3381 and 25.2979 Å were observed. These results characterize form I of nevirapine [26]. Flowability is an important factor in several processes in the pharmaceutical industry, including the compression process. Fig. 3 shows the results for the apparent and compacted density. A reduction in the volume of the powder was observed soon after the start of the test, and the volume stabilized after 5 min. The use of 2000 taps is usually recommended for measurements of the compacted density; this number was obtained by checking for changes in volume during tests until there was stabilization of the observed volume [27]. The result for the density of the compacted powder in our tests was 0.5748 g/cm3. The compressibility index CpI is related to the accommodation of the particles [28]. The result for the Hausner ratio was 1.45, and that for the Carr index was 31.24%. A Hausner ratio below 1.25 indicates good compressibility. The Carr index may be indicative of the flowability and degree of packing of a material, which are relevant properties when the matrices of a tableting machine are being filled. A Carr index lower than 15% indicates an adequate flow of the powder and stable packing, whereas values above 25% are characteristic of poor flow properties [29].
599
To improve flowability, changes in the crystal structure or in the particle size have been suggested, these being criteria for comparison between different batches of raw material. The data obtained with the helium pycnometer are shown in Table 1. The volume and true density obtained for the nevirapine were 0.1365 cm 3 ± 0.009329 and 1.6170 g/cm 3 ± 0.0008%, respectively. There is a significant difference between this value for the density and the data presented earlier for the compacted density of the same material. The difference between the two data sets is related to how they were obtained. The compacted density takes account of the total volume occupied by the powder. Although the particles are in contact, however, empty spaces still exist between the particles even after the compaction operation. The data from the pycnometer are obtained from the difference between the volumes occupied by the inert gas (He) in the crucible container with and without the powder. The empty space in the powder is occupied by the gas, and the true density of the drug is determined from the difference in volume. Solubility is an important parameter both for the in vitro evaluation of solid dosage forms and for proper dissolution and subsequent absorption in vivo. According to the BCS, for a drug to be considered highly soluble, a dose/solubility value of less than 250 mL is necessary over the whole pH range to which the drug will be exposed in the gastrointestinal tract. Data obtained, after equilibration, for the solubility of the drug in various buffer media are shown in Fig. 4. According to the solubility data and based on the usual dose of the drug (200 mg), it can be estimated that only the conditions present in the stomach (pH b 2) will be sufficient for the complete dissolution of nevirapine. The dose/solubility value in other media is greater than 250 mL. According to the BCS, this value defines the drug as poorly soluble, and, possibly, the step of dissolution of the drug from the dosage form may interfere with its bioavailability [30,31]. From Fig. 5, it can be seen that nevirapine is sensitive to pH, and this is one of the factors contributing to its dissolution. In an acid medium containing >2.10 g/L [H3O+], the solubility of the drug was 4.99 g/L. When the environment was changed using solutions with different pH values, the solubility of the drug was reduced by a factor of 10–40. Structural changes in the drug molecule in solution are responsible for the different solubility values at different pH values. The electron donor sites (nucleophiles) in nevirapine structure, represented by the pyridine nitrogen atoms, can be protonated under acidic conditions, forming a stable zwitterion form. By reducing the concentration of H3O+ ions, this form tends to stabilize the molecule [32]. Aungst et al. [33] studied an antiretroviral drug (DPC 961) of low solubility and high permeability in vitro; the dissolution was clearly impaired in media with pH values from 4.5 to 7.2. However, data obtained after administration of pharmaceutical formulations showed
H
H N
+
N
N
+
NN
N
N
+
[H 3 O ]
+ N H O
CH 3
CH 3
O
H2O
H
H N
+
N
N
+
N -O
CH 3
Fig. 5. Diagram representing the balance of molecular and protonated forms of nevirapine in aqueous medium.
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 the drug. Thus, a dissolution test employing a surfactant concentration similar to that found under normal conditions in the TGA may be one way to evaluate and obtain results that are more representative of the likely behaviour of the drug in vivo. To study the impact of the presence of surfactants on the solubility of nevirapine, assays were performed using phosphate buffer solutions with sodium lauryl sulphate added at concentrations of 0.1, 0.5 and 1.0% (w/w). This methodology was employed to reduce the time and cost requirements of the data acquisition and quantification method. However, the presence of other constituents in the medium that mimic the gastrointestinal tract (e.g. lecithin and bile salts) would be expected to favour the solubilization and subsequent absorption of the drug. One negative aspect of the employment of surfactants is their impact on the method of quantification, especially because they might interfere with the UV analysis. In the solubility tests, the quantification of the samples containing the drug in the presence of surfactant was 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 sodium lauryl sulphate (Fig. 6) showed that even at high pH (> 4.5), under which conditions the drug has low molecular dissolution, the presence of an additional compound to reduce the surface tension and increase interaction with hydrophobic regions of the molecule favoured the diffusion of nevirapine in the liquid phase. Using only the solubility data for pH = 6, in which the pH is a critical factor, we would expect that dissolution in the normal gastrointestinal tract would most likely occur when the drug was precipitated by alkalinization of the stomach contents at the entrance to the duodenum, and this is a factor critical for absorption. However, even the highest concentration (1%) of anionic surfactant used in the solubility measurements still does not generate surface tension values similar to those produced by bile salts and cholesterol in vivo. Thus, it is likely that the solubility of the drug is greater in the gastrointestinal tract [34]. The speed at which a solid dissolves in a dissolution medium or a biological fluid is a function of its surface area, solubility and dissolution rate constant, and of the solute concentration in the medium. Because of this, it is appropriate to study the behaviour of a drug during the dissolution process by carrying out the experiment in such a way that the surface area is kept constant. This can be done by employing an apparatus for measuring the intrinsic dissolution rate. The IDR of nevirapine was 0.4109 mg/cm 2 min, according to a linear regression of the data obtained in the test (y = 0.4109x + 0.4059; R 2 = 1). The data obtained from the determination of the IDR can provide information about the behaviour of the drug prior to absorption, since dissolution is a continuous phenomenon, with differences in solubility, and these data can be better correlated with the dynamics of dissolution in the gastrointestinal tract.
4
Nevirapine
2
Heat Flow (W/g)
600
Nevirapine+Lactose
0 -2
Nevirapine+Methocel K4M
-4
Nevirapine+Kollidon VA64
-6
Nevirapine+Kollidon K30
-8
Nevirapine+Cellulose MC
-10 -12 0
50
100
150
200
250
300
350
Temperature (ºC) 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 and β = 10 °C/min.
A drug may be considered highly soluble if it has an IDR above 1.0 mg/cm 2 min; in this case, bioavailability problems are less common [35]. However, values of the IDR below 0.1 mg/cm 2 min indicate that bioavailability problems may arise because of drug-dependent dissolution. When the data obtained from the IDR tests are compared with the data from the measurements of the solubility in acid (0.1 N HCl), we can see that the dissolution test was carried out under sink conditions, i.e. the maximum concentration of the drug was lower than 15% of the solubility in the dissolution medium. Thus, the low intrinsic dissolution value found was not influenced by saturation of the medium. The IDR varies from drug to drug and may be related to the diffusion coefficient and the thickness of the film formed between the solid surface and the liquid medium. If different batches of the same drug are tested under the same conditions, the value of the intrinsic dissolution constant will be affected only by factors inherent to the drug, such as the method of synthesis, crystallinity, particle size and polymorphism [36]. To study possible physical interactions and collect data to provide evidence of any incompatibility, nevirapine was evaluated by DSC and TGA both in the form of the pure drug and in the form of a 1:1 physical mixture of the drug with an excipient. The excipients used were Kollidon® VA64, Kollidon® K30, hydroxypropylmethylcellulose, microcrystalline cellulose PH 101 and lactose. The thermoanalytical curve obtained from DSC shows that the drug has 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 related to 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 110
6
Weight (%)
5
Solubility (g/L)
Nevirapine Nevirapine+Lactose Nevirapine+Methocel k4M Nevirapine+Kollidon VA64 Nevirapine+Kollidon K30 Nevirapine+Celulose MC
90
4 3
70 50 30 10
2 -10 1
-30 0
0 HCl 0.1 N
HCl 0.01 N
Buffer pH 6
Buffer pH 6 + Buffer pH 6 + Buffer pH 6 + 0.1% 0.5% 1.0%
Fig. 6. Solubility of nevirapine (g/L) in various buffer solutions, and in buffer solutions added with 0.1, 0.5 and 1.0% sodium lauryl sulphate.
200
400
600
800
Temperature (ºC) Fig. 8. Thermogravimetric curves of nevirapine and physical mixtures in 1:1 (w/w) of excipients used in the compatibility test. Analyses were performed under inert atmosphere (N2) 50 mL/min, m= 10.0 ± 0.1 mg and β = 10 °C/min.
G.G.G. de Oliveira et al. / Materials Science and Engineering C 33 (2013) 596–602 Table 2 Thermodynamic data obtained from the thermoanalytical curves DSC, TG and DTG of mixture between nevirapine and excipients. Nevirapine/excipients
tmelting (°C)
tonset TG
1° tpeak DTG
2° tpeak DTG
Nevirapine Nevirapine + cellulose MC Nevirapine + lactose Nevirapine + PVP K30 Nevirapine + PVP VA 64 Nevirapine + Methocel K4M
246.48 246.54 239.44 237.17 233.52 242.63
286.03 258.48 257.92 281.11 282.07 282.07
250.37 249.13 243.09 247.56 254.94 248.16
329.56 300.48 314.09
a
a
305.33 318.89
Non-observed peak due to overlapping.
was reached. The thermoanalytical curve was also used to estimate the purity of the drug, using the cryoscopic equation provided by the TA Universal Analysis programme. The purity was found to be 99.0% or greater; however, the correction had a high value (20%). The adequacy of the method for estimating the purity is highly dependent on the correction, which should be below 10% to provide reliable data. The use of a lower weight and lower heating rate may be the reason for the 20% value. DSC curves of the mixtures of nevirapine with the various excipients are shown in Fig. 7. In general, when a mixture of an excipient and a drug is employed, interaction may occur, and the physical properties of the mixture and the dosage can be seriously affected. If there is no interaction, either physically or chemically, then the mixture should show the same thermal properties as the individual products [37]. The TGA profile was also obtained (Fig. 8). The thermogravimetric curve indicates that the total mass loss occurs in only one event, which starts from about 200 °C with tonset = 271.41 °C and extends up to 320 °C. A mathematical treatment of the data using differentiation indicates 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%; the peak is at 246.98 °C, extending to 256.88 °C with a residual mass of 87.63%. The second peak has tonset = 306.30 °C and a residual mass of 20.87%; the peak is at 315.69 °C, extending to 323.74 °C with 0.02% residual 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) and cooling rate of 10 °C/min (β). Thermodynamic data for nevirapine obtained from the DSC, TGA and differential thermogravimetry (DTG) thermoanalytical curves are presented in Table 2. When we observe the thermoanalytical profiles of pure nevirapine and of the binary mixtures with the excipients, the endothermic melting event characteristic of the drug is present in all of the DSC curves. The microcrystalline cellulose and lactose mixtures show the characteristic melting peak of nevirapine, but with a clear shift relative to the initial run. The mixtures with Kollidon® VA64, Kollidon® K30 and Methocel® K4M do not show the characteristic melting peak of nevirapine; there is only a minor thermal event, shifted to the left and with a reduced enthalpy. It is clear that all of the excipients, with the exception of microcrystalline cellulose, interact with the drug by changing its thermoanalytical profile. The DSC analysis of lactose usually shows three endothermic events due to the two most common forms of lactose, at 140, 220 and 240 °C, corresponding to the dehydration of α-lactose and the melting of α- and β-lactose, respectively. When the
601
mixture is subjected to heating (Fig. 7), only one event is observed in the temperature range 225–250 °C. Apparently, the melting of lactose causes a displacement of the melting peak, promoting drug dissolution and partial melting at a lower temperature. In general, this is not indicative of any potential incompatibility. Nevertheless, TGA was carried out to provide data on the chemical and structural changes in order to clearly characterize this event (Fig. 8). The resulting thermograms of the mixtures are sums of the events related to the two components, including the degradation reaction and the mass loss involved. The data obtained from the TGA curves have been summarized above. The parameters used for the comparison of the thermoanalytical curves were the melting temperature, the onset temperature of weight loss (tonset TGA) and the temperature of maximum rate of mass loss (a typical TGA 1 and 2 event). The decrease in tonset TGA was attributed to the presence of lactose in the binary mixture. The TGA peak was 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, indicating only physical interaction between these excipients and the drug. This phenomenon was also reported by Grangeiro et al. [38] when PVP was used; it was explained by the formation of crystalline microaggregates of the drug and dispersion in a matrix of this amorphous polymer. The evaluation of a drug for degradation under light exposure should employ tests that allow degradation products and/or structural changes of the drug molecule to be identified [39]. Samples (pure nevirapine and 1:1 physical mixtures with the excipients) were exposed to light before further DSC and TGA analysis (Figs. 7 and 8). The powders were evaluated for changes in appearance in comparison with the product stored under suitable storage conditions. After the exposure period, there were no changes in the colour of either the drug powder or solutions of it, where precipitation was also not observed. The data obtained from DSC analysis showed no changes in the event related to the melting of the drug (at 240 and 250 °C). The peaks related to this event showed similar endothermic peaks (at 246.58 and 246.23 °C) for samples of nevirapine before and after exposure to light. The TGA data confirmed the absence of changes. The overlapping curves indicated that there was no impact on the events related to mass loss or on the peaks related to the derivative, indicating that neither the temperature at which degradation occurs nor the rate at which degradation occurs had changed. The samples were subjected to further quantification to assess the impact of light on the samples in solution. The results are shown in Table 3. The data showed that light exposure did not cause severe degradation of the drug in aqueous media. However, in acid solutions, a decrease in the drug concentration was observed compared with the samples in phosphate buffer media at two different pH values. After light exposure, the nevirapine was analysed further by DSC and TGA (Figs. 9 and 10). 4. Conclusions Physicochemical analysis has been performed to obtain information about nevirapine prior to formulation. The results show that
Table 3 Data obtained after quantification of the drug nevirapine in different buffer solutions using spectrophotometric method. Readings were taken at λ=294 nm and 313 nm (0.01 N HCl). The data presented were obtained in control through the use of solutions filled into ampoules with similar feature used in the other samples but covered with aluminium. Solution
HCl 0.1 N HCl 0.01 N Buffer 0.1 pH 4.5 Buffer 0.1 pH 7.2
Control
0.1945 0.2188 0.2071 0.2070
Readings
0.1941 0.2172 0.2064 0.2055
Average
0.1932 0.2197 0.2070 0.2068
C = concentration control solution (μg/mL). A= concentration sample (μg/mL).
0.1937 0.2179 0.2077 0.2071
0.1937 0.2183 0.2070 0.2065
Concentration (μg/mL) C
A
18.83 19.37 19.98 19.97
18.75 19.32 19.98 19.92
% of recovery
99.59 99.76 99.97 99.76
602
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3
Superior) and FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo).
Nevirapine Heat Flow (W/g)
2
References
Nevirapine photostability 0 -2 -3 -5 -6 0
100
200
300
400
Temperature (ºC) Fig. 9. DSC of nevirapine before and after light exposure. The curve was performed under inert atmosphere (N2) 50 mL/min, m ≈ 2.0 mg and β = 10 °C/min.
0.10
140
0.00
Weight (%)
100 80
-0.10
60 Nevirapine
40
-0.20
Nevirapine TGA
20
Nevirapine photo
0
-0.30
2nd Deriv. weight (%/ºC^2)
120
Nevirapine photostability TGA
-20
-0.40 0
100
200 300 400 Temperature (ºC)
500
600
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.
the molecule has high stability in the solid state and is not susceptible to degradation under light exposure. It has a high melting point (246 °C), which is the only event in the DSC profile, and low hygroscopicity, facilitating handling of the drug in the normal environment. An investigation of its interaction with other excipients (lactose, microcrystalline cellulose, PVP/PVA and HPMC) selected for their wide use in solid dosage forms showed no incompatibilities. The drug has low solubility, an acid medium being the most appropriate medium for assessing the release profile of solid dosage forms. From our tests, dissolution was shown to be the critical factor affecting the bioavailability; this is typical of a class II drug according to the BCS.
Acknowledgements The authors wish to acknowledge support from the Fundação para a Ciência e Tecnologia do Ministério da Ciência e Tecnologia under reference number ERA-Eula/0002/2009. Ms Severino received sponsorship from CAPES (Coordenação Aperfeiçoamento de Pessoal de Nivel
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