DOI: 10.1021/cg901501d
Influence of the Composition of Water/Ethanol Mixtures on the Solubility and Recrystallization of Nevirapine
2010, Vol. 10 3859–3868
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Nicole Stieger,*,† Mino R. Caira,‡ Wilna Liebenberg,† Louwrens R. Tiedt,§ Johanna C. Wessels, and Melgardt M. De Villiers^ †
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Unit for Drug Research and Development, Faculty of Health Sciences, North-West University, Potchefstroom, South Africa, ‡Department of Chemistry, University of Cape Town, Rondebosch, South Africa, §Laboratory for Electron Microscopy, Faculty of Natural Sciences, North-West University, Potchefstroom, South Africa, Pharmaceutical and Biomedical Services, Faculty of Health Sciences, North-West University, Potchefstroom, South Africa, and ^School of Pharmacy, University of Wisconsin, Madison, Wisconsin Received December 1, 2009; Revised Manuscript Received July 8, 2010
ABSTRACT: When anhydrous nevirapine was recrystallized from ethanol that contained increasing amounts of water, it was found that the amount of water affected not only the solubility but also the solvent inclusion and crystal habit of the precipitated material. This led to the crystallization of a previously unknown nevirapine hemiethanolate from solutions that contained less than 5% water. Adding more than 10% water led to the isolation of the known hemihydrate, and a further increase in watercontent caused precipitation of the anhydrous form. It was shown not only that discontinuities in the solubility profile indicate transition from one crystal form to another but also that they occurred at the junction between two distinct crystal forms or crystal habits. A possible explanation for these observations was found in the relationship between the solubility of nevirapine, the solubility parameter of the solvent mixtures, and the change in water activity.
Introduction Nevirapine is a dipyridodiazepinone antiretroviral used in the treatment of HIV-1 infection and AIDS. It is a non-nucleoside reverse transcriptase inhibitor (NNRTI) with the systematic name 11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[3,2-b: 20 ,30 -e][1,4]diazepin-6-one. It is of particular importance in preventing mother-to-child HIV transmission. To minimize viral resistance, this drug is almost exclusively used in combination therapy.1,2 Although complete solubility and permeability data for nevirapine are lacking, the FDA classifies it as a Class II (high permeability, low solubility) drug.3 By virtue of its weakly basic character and ionization (pKa 2.8), nevirapine exhibits pH dependent solubility.4 At pH values less than the pKa, it is highly soluble in aqueous buffer. Nevirapine is administered orally in the form of tablets (anhydrous form) or suspensions (hemihydrate). Its bioavailability decreases at higher doses due to absorption being solubility rate-limited.5 Apart from its use in the treatment of HIV/AIDS, nevirapine also exhibits anticancer activity. Endogenous reverse transcriptase (RT) is emerging as a novel molecular target in cancer therapy because its inhibitors, including nevirapine, exhibit a differentiating activity in human tumor cells which is mediated by their ability specifically to reprogram gene expression, thereby restoring cell function lost during tumor progression.6,7 This hypothesis has recently been substantiated in a patient affected by a poorly differentiated thyroid carcinoma, when nevirapine restored the uptake of radioactive iodine and thyroglobulin and sodium-iodide symporter (NIS) expression in tumor cells.8 The drug has also been tested against prostate and cervical cancer.9,10
However, while nevirapine is a safe treatment in immunocompromised patients, nevirapine-containing regimens have been associated with severe immune-mediated toxicities in individuals not infected by HIV.11 Potential toxicity might contradict the use of conventional oral delivery systems (capsules, tablets, and suspensions) for treating cancers with nevirapine. Solutions of this poorly water-soluble drug in cosolvent systems such as ethanol/water mixtures might be an alternative delivery approach. Such solutions can potentially be used to formulate injectable forms of nevirapine for local administration directly into cancerous tissue.12 In this study we report the solubility of nevirapine in ethanol/ water cosolvent mixtures. We chose ethanol because intratumoral injection of absolute ethanol is an established technique for the treatment of many types of cancer, including hepatocellular carcinoma, 13 esophageal cancer, 14 renal tumors,15 and tracheobronchial lesions.16 The ethanol causes necrosis of cancerous cells but leaves the surrounding healthy tissue and vasculature intact. As such, we do not anticipate any unforeseen adverse effects to result from a high ethanolcontent nevirapine injection to be used for intratumoral administration. Since solutions of poorly water-soluble drugs tend to be unstable, we also studied the crystallization of nevirapine from these solutions. In particular, we tried to correlate crystal form and habit changes to changes in solvent properties such as solubility parameter and water activity. Materials and Methods
*Corresponding author. Private Bag X6001, Unit for Drug Research and Development, North-West University, Potchefstroom, 2520 South Africa. E-mail:
[email protected]. Telephone: þ27 18 299 4357. Fax: þ27 18 293 5219.
Nevirapine (anhydrous) was purchased from Cipla (Mumbai, India, batch number: 1001003). Analytical grade ethanol (min. 99.5% v/v, max. 0.2% water), used as recrystallization medium, was obtained from Saarchem (Wadeville, South Africa, batch number: 1030779). All water used was prepared with a Millipore Milli-Q Ultrapure Water Purification System (USA). The recrystallization procedure was as follows: 2 g of anhydrous nevirapine was weighed into each of ten glass beakers, and 90 mL of
r 2010 American Chemical Society
Published on Web 08/06/2010
pubs.acs.org/crystal
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Figure 1. Solubility diagrams for nevirapine in mixtures of ethanol and water: (A) solubility (mg/100 mL at 25 C) versus the percentage (v/v) of water added to ethanol; (B) diagram taking into account the volume increase as antisolvent is added (concentration (mg/volume at 25 C) versus the percentage of water added to 90 mL of ethanol); (C) degree of saturation of the recrystallization medium once it has cooled to 25 C. ethanol was added. Previous experiments showed the approximate maximum solubility of nevirapine, in ethanol at 75 C, to be 1 g per
45 mL. The nevirapine was dissolved by heating and agitation on Heidolph (Germany) MR3001K magnetic stirrers. Once the solutions
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Table 1. Summary of the Influence of Ethanol/Water Composition on the Crystal Forms and Crystal Habits of Nevirapinea recrystallization medium EtOH/H2O (mL)
approximate water content (% v/v)
90:0 90:4.7 90:10
0 5 10
90:22.5 90:38.6 90:60 90:90 90:135 90:210 90:360
20 30 40 50 60 70 80
a
nevirapine form obtained
crystal habit
hemiethanolate hemiethanolate mixture of anhydrous, hemiethanolate, and hemihydrate hemihydrate hemihydrate hemihydrate hemihydrate hemihydrate hemihydrate anhydrous
tabular crystals smaller tabular crystals very small tabular and columnar crystals disorganized columnar clusters disorganized columnar clusters columnar clusters platy clusters platy crystals with rudimentary clustering platy crystals very small bladed crystals
The amount of anhydrous nevirapine dissolved in each solvent system was 2 ( 0.01 g throughout.
Figure 2. Morphology of the hemiethanolate of nevirapine: (A) tabular crystals as seen through a stereo light microscope; (B) SEM image of the hemiethanolate obtained from ethanol only; (C) SEM image of the hemiethanolate recrystallized from ethanol containing 5% water. were clear, increasing volumes of water, viz. 0, 4.7, 10, 22.5, 38.6, 60, 90, 135, 210, and 360 mL, were added gradually to the respective beakers. The latter were then removed from the magnetic stirrers and covered, and the solutions were left to cool at ambient temperature. The time required for crystal growth varied: minutes (80% v/v or more water), hours (0%, 5%, 10%, 60%, and 70% v/v water), or days (20%, 30%, 40%, and 50% v/v water). Crystals were harvested from the mother liquor and dried briefly on filter paper prior to analysis. Samples were analyzed using the following: • Differential scanning calorimetry (DSC): Shimadzu DSC-60A (Japan) with TA60 version 2.11 software. Approximately 2-4 mg of each sample was weighed and heated in closed aluminum crucibles. Samples were heated at 10 K/min in an inert nitrogen atmosphere. • Thermogravimetric analysis (TGA): Shimadzu DTG-60 (Japan) with TA60 version 2.11 software. Samples were heated from 25 to 300 C at 10 K/min, in open aluminum crucibles. Nitrogen gas was used as inert atmosphere. • Fourier transform infrared spectroscopy (FT-IR): Shimadzu IRPrestige-21 (Japan), using a Pike Multi-Reflectance ATR accessory, with Shimadzu IRsolution version 1.40 software.
• •
• • •
Spectra were recorded over a range of 400-4000 cm-1. KBr was used as background. The sample was dispersed in a matrix of powdered potassium bromide and, through diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), the IR-spectrum was measured in a reflectance cell. X-ray powder diffraction (XRPD): PANalytical XPert-Pro (Netherlands). Karl Fischer titration: Metrohm 870 KF Titrino Plus (Switzerland) autotitrator. The titrator was calibrated using a predetermined mass of water (25-30 μL) and Hydranal water standard 10.0. Approximately 50 mg of each sample was used for the moisture determination. The experiment was performed in duplicate for each sample and the average value used. UV spectroscopy: Shimadzu UV-1800 (Japan). Solubility was determined using the method described by Stieger et al.17 Scanning electron microscopy (SEM): FEI Quanta 200 ESEM. The samples were coated with Au/Pd, to a thickness of 20 nm, and the ESEM operated at 10 kV under high and low vacuum modes. The single crystal X-ray structure of the new hemiethanolate was determined using the same procedures reported earlier.18 Due to the exceptional instability of this solvate, the single crystal
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Figure 3. DSC traces of the nevirapine forms recrystallized from various ethanol/water mixtures: (A) hemiethanolate; (B) mixture of forms from H2O/EtOH 90:10; (C) hemihydrate; (D) anhydrous. The onset temperatures for desolvation and fusion are indicated, as well as the peak temperatures for desolvation. selected for analysis was fully coated with oil directly after its removal from mother liquor in order to minimize desolvation. The crystal was then immediately placed in the cold nitrogen stream on the diffractometer and cooled to -100 C to reduce atomic thermal vibration in order to optimize the location of the guest ethanol molecules. X-ray crystallographic information for the crystal structure of 1 has been deposited with the Cambridge Crystallographic Data Centre, CCDC No. 756353. DSC, TGA, and KF results were rounded to the nearest reproducible decimal. The precision of the melting points recorded in the DSC experiments, based on multiple measurements, is (0.05 C. Both KF and TGA average weight percentages conformed to a relative standard deviation of 2% or less.
Results Using the method described by Stieger et al.,17 the solubility of anhydrous nevirapine, in each of the binary solvent systems, was determined by UV spectrophotometry. Results of the solubility determination are given in Figure 1. The relative standard deviation for solubility values was 1.7% or less. Although nevirapine is ∼80 times more soluble in ethanol (8 mg/mL) than in water (0.1 mg/mL), the maximum solubility (16 mg/mL) was measured in a 80:20 v/v ethanol/water mixture. Figure 1A and B shows the relationship between solubility and solvent composition and the effect that relationship has on the crystal form obtained. Figure 1C seems to indicate that dramatic changes in the degree of saturation can be associated with a change in crystal habit, even if no change in crystal form occurs. Table 1 gives a summary of the composition of recrystallization media and corresponding crystal forms/habits recovered. Although nevirapine is available predominantly as the anhydrous form or a hemihydrate, it recrystallized from ethanol, containing 5% or less water, as a novel hemiethanolate. The crystals obtained were tabular, with the only difference between those obtained from solutions containing 0% and 5% water being that the latter were approximately 50% smaller. As can be seen in Figure 2B and C, the crystals that were well-defined in part A cracked and fractured during SEM analysis. This indicates that desolvation occurred due to the vacuum that was applied to the sample. The DSC trace (Figure 3) of this crystal form shows desolvation at 92.5 C
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Figure 4. XRPD patterns for three forms of nevirapine and for the mixture of forms obtained from ethanol containing 10% water.
and a melting endotherm at 245.5 C. A 7.9% weight loss, determined by TGA, corresponds with the theoretical 7.4% loss one would associate with a hemiethanolate. Karl Fischer titration confirmed the absence of water. The XRPD pattern (Figure 4) of this form also differed from those of both the hemihydrate and anhydrous form;it shows a distinct peak at 7.3 2θ which is not present for either of the other two forms. The FT-IR spectrum (Figure 5) displays strong O-H stretching bands in the 3700-3584 cm-1 region, as is typical with alcohols, indicating the possible inclusion of ethanol in the crystal structure. The inclusion of ethanol was also confirmed by single crystal structure analysis. Crystallographic and refinement details are listed in Table 2. The crystallographic asymmetric unit, comprising one nevirapine molecule and half of an ethanol molecule, is shown in Figure 6. The trial structure employed in the solution of 1 was the set of non-H atoms of the nevirapine molecule occurring in the isostructural ethyl acetate solvate.18 Following anisotropic least-squares refinement of these atoms, H atoms were located in difference electron density syntheses and were added in idealized positions in a riding model with isotropic thermal parameters in the range 1.2-1.3 times those of their parent atoms. Four significant residual difference electron-density peaks appeared. These were interpreted as two disordered ethanol half-molecules (C23-C22-O21 and C22-C23O24, Figure 6), each with site-occupancy factors (s.o.f.s) of 0.25, consistent with the 2:1 nevirapine/ethanol stoichiometry determined by TGA. The carbon atoms (common to both guest orientations) therefore have s.o.f.s of 0.5, while each oxygen atom has s.o.f. 0.25. Due to the disorder, no H atoms were included in the solvent modeling and the common variable isotropic thermal parameter of the C and O atoms was refined to 0.152(4) A˚2, indicating considerable thermal motion, even at the relatively low temperature of the analysis. Distance constraints of 1.54 and 1.45 A˚ for the C-C and C-O bonds respectively were applied to maintain reasonable molecular geometry. The crystal structure comprises a framework of centrosymmetric nevirapine dimers, assembled in such a way as to provide infinite channels parallel to the b-axis for solvent molecule accommodation (Figure 7). Centrosymmetric host dimers are formed via N-H 3 3 3 O hydrogen bonding with N 3 3 3 O 2.923(4) A˚ and angle N-H 3 3 3 O 175(1). The threedimensional assembly of nevirapine molecules is identical to
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Figure 5. FT-IR spectra for the anhydrous (D), hemihydrate (C), and hemiethanolate (A) forms of nevirapine and for the mixture (B) of forms obtained from ethanol containing 10% water.
that reported earlier for the isostructural hemisolvates of nevirapine with ethyl acetate and dichloromethane.18 Location of these volatile solvents in infinite channels accounts for the spontaneous desolvation of their respective crystal solvates when the latter are exposed to air. The moderate precision attained in the X-ray analysis of 1 (reflected in relatively high R-factors for a structure of this size) stems from the disorder and abnormally high thermal vibration of the included solvent molecules. Analogous solvates of nevirapine under current investigation show similar trends, despite the apparently high optical uniformity and excellent morphology of their crystals. To establish that the reported crystal structure is representative of the bulk phase, the single crystal X-ray data for 1 were used to calculate its XRPD pattern (Figure 8). The overall profile is in good agreement with the experimental trace (Figure 4, top). Spontaneous partial desolvation during recording of the experimental trace at room temperature was inevitable and partly accounts for discrepancies in peak intensities. Relative to the experimental pattern, peak positions in Figure 8 are slightly shifted to higher angles due to the low temperature of the X-ray analysis. The experimental and calculated XRPD pattern was also compared with those generated for a series of nevirapine solvates whose structures we reported earlier.18 From this comparison, it was evident that the hemiethanolate is isostructural with both the ethyl acetate and dichloromethane hemisolvates. While the primary focus of this study is the variety of nevirapine crystal phases originating from ethanol/water
mixtures, their domains of crystallization, their morphologies, as well as the solubility of nevirapine in said mixtures, it should be noted that the X-ray analysis of 1 reported here reveals the principal structural features of this novel solvate that could explain some of the crystal form changes observed during the solubility measurements. For example, it was found that, for the hemiethanolate 1, guest exchange on immersion of the crystal in water presumably involves initial displacement of ethanol molecules in the channels by incoming water molecules. However, prolonged storage of the hemiethanolate crystals results in a solution-mediated phase transformation, with the final product being the stable crystalline hemihydrate, with a different (monoclinic) crystal structure. To understand the stability and transformation of the hemiethanolate, an alternative method of recrystallization was employed in an attempt to slow down growth and improve the quality and size of the crystals yielded. Instead of allowing the nevirapine solution to cool to ambient temperature, the beaker was covered and placed in an oven for 3 days. The temperature was initially set at 60 C and was gradually decreased until it reached 25 C on the last day. This method of recrystallization produced larger well-defined single crystals, but they proved to be the anhydrous form of nevirapine. It seems that the high rate of crystal formation and growth, observed with the original method of recrystallization, is essential to ensure solvent inclusion. It follows that, should it be necessary to produce large batches of hemiethanolate, the larger volume of mother liquor, and associated slower cooling rates, will necessitate the use of active cooling
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Table 2. Crystal and Refinement Parameters for the Hemiethanolate of Nevirapine 1 ratio empirical formula fw T (K) wavelength (A˚) cryst syst space group a (A˚) b (A˚) c (A˚) R (deg) β (deg) γ (deg) V (A˚3) Z dcalcd (Mg/m3) abs coeff (mm-1) crystal size (mm3) θ range for data collection index ranges no. of reflections collected no. of independent reflections completeness (%) abs corr max. and min. transmission refinement method data/restraints/parameters GOF on F2 final R indices [I > 2σ(I)] R indices (all data) largest diff peak and hole, e A˚-3
2:1 nevirapine/ethanol C15H14N4O 3 (C2H6O)0.5 289.34 173 ( 2 0.71073 triclinic P1 7.8809(16) 8.4738(17) 12.428(3) 85.17(3) 90.01(3) 67.25(3) 762.2(3) 2 1.261 0.084 0.10 0.12 0.13 3.00-25.68 -9 e h e 9 -10 e k e 10 -15 e l e 15 10054 2758 [R(int) = 0.0656] 96.1 none 0.989 and 0.992 full-matrix least-squares on F2 2758/5/195 1.037 R1 = 0.0867, wR2 = 0.2179 R1 = 0.1290, wR2 = 0.2548 0.79 and -0.40
Figure 7. Perspective view of the structure of the hemiethanolate of nevirapine showing the ethanol-filled channels defined by the framework of nevirapine dimers.
Figure 8. Computed XRPD pattern of solvate 1 (Cu KR radiation).
Figure 6. Crystallographic asymmetric unit in 1, the hemiethanolate of nevirapine. Thermal ellipsoids for the drug molecule are drawn at the 50% probability level. Atoms of the disordered ethanol half-molecule are represented as spheres with a common isotropic thermal parameter.
strategies. In the laboratory setting, it was found that this could be achieved easily for volumes up to 2000 mL by simply placing the covered receptacle in a refrigerator at 5 C ((2 C). When the recrystallization mixture has the composition H2O/EtOH 90:10, the resulting crystals are a mixture of the hemihydrate, hemiethanolate, and anhydrous forms. SEM photographs show very small but well-defined crystals that range from columnar to tabular (Figure 9). DSC analysis (Figure 3) records desolvation starting at 82.4 C, with twin
Figure 9. SEM image of the mixture of forms obtained from ethanol containing 10% water.
endotherms peaking at 90.0 and 127.0 C;this suggests the possible presence of two solvents. The XRPD trace (Figure 4) has the same peak as the hemisolvate at 7.2 2θ but also displays the characteristic peak for the hemihydrate at 5.6 2θ
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Figure 10. SEM images of the different crystal habits of nevirapine hemihydrate as influenced by the amount of water introduced to the ethanol from which it was recrystallized. Water added: (A) 20%; (B) 30%; (C) 40%; (D) 50%; (E) 60%; (F) 70%.
and the peak at 9.5 2θ that is associated with both the hemihydrate and anhydrous forms. The FT-IR spectrum (Figure 5) resembles that of anhydrous nevirapine, but it also shows the presence of O-H groups. Due to the very small particle size and the fact that the moist crystals form a malleable paste, it was impossible to adequately dry samples for TGA and Karl Fischer titration without completely desolvating the hemiethanolate component. The ratio in which the three forms are present is therefore not known. It has been noted that the hemiethanolate component undergoes solvent exchange over time. When the mixture was left in the mother liquor for a period of one month, the hemiethanolate transformed to the hemihydrate. FT-IR analysis showed that the anhydrous component was still present. The addition of 20-70% water to the ethanol/nevirapine solution results in the formation of the hemihydrate form. However, the crystal habit of the hemihydrate is affected by the amount of water added (Figure 10). If 20-40% water is
present, clusters of columnar crystals form. The clusters become progressively more organized as the amount of water increases. With 50% water, clusters consist of platy crystals. In the presence of 60% water, platy crystals form and some rudimentary clusters are also present. Organization is lost with 70% water, and no more clusters are evident;only individual platy crystals. The DSC traces (Figure 3) for these six recrystallization products all correspond with the known hemihydrate form,19 as do the XRPD patterns (Figure 4) and FT-IR spectra (Figure 5). The amount of water present for each product was confirmed with TGA and Karl Fischer titration and closely matches the theoretical 3.2% expected for a hemihydrate. When 80%, or more, water was added, precipitation was almost instantaneous;the solution turned opaque immediately and small crystals (Figure 11) formed within minutes. DSC (Figure 3), XRPD (Figure 4), and FT-IR (Figure 5) confirmed the product to be the known anhydrous form of nevirapine.19
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Figure 11. SEM image of the bladed crystals of anhydrous nevirapine produced when 80% water is added to the saturated solution.
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Figure 12. Change in solubility (mg/mL at 25 C) of nevirapine as a function of the change in magnitude of the solubility parameter of the ethanol/water mixtures.
Discussion In this experiment, ethanol was chosen as “solvent” because of nevirapine’s relatively high solubility in it. Water was the “anti-solvent”, because nevirapine is extremely hydrophobic and poorly soluble in neutral pH aqueous media. As is typical for systems prone to solvate formation,20 solubility did not decrease smoothly as increasing amounts of the antisolvent were added (Figure 1). Instead, the solubility versus solvent composition of nevirapine is a complex relationship with several discontinuities. Byrn et al.20 state that these discontinuities demarcate the boundaries between two crystal forms. While our results can confirm this, Figure 1 also shows the presence of discontinuities where changes in crystal habit occur. Therefore, it might be said that a discontinuity in the relationship of concentration versus binary solvent composition represents a boundary between either different crystal forms or different crystal habits. The solubility profile of nevirapine in the ethanol/water cosolvent system is complex, with multiple crystal and habit changes, suggesting that the nature of the cosolvent and its interaction with water also play an important role. One way to quantify the observed changes is to calculate the solubility parameters of each ethanol/water mixture. In 1936, Joel H. Hildebrand introduced the concept of a “solubility parameter” (δ) as a way to quantify solvent properties.21 Hansen extended the Hildebrand parameter method to estimate the relative miscibility of polar and hydrogen bonding systems. In Hansen’s approach, the Hildebrand solubility parameter (δ) is split into three components: polar (δp), dispersion (δd), and hydrogen bonding (δh); thus, the name 3D solubility parameters.22 δ2 ¼ δd 2 þ δp 2 þ δh 2 For a solvent blend, such as an ethanol/water mixture, the hydrogen bonding component is calculated from the solubility parameters of the pure solvents and their volume fraction in the mixture. In Figure 12 the change in solubility as a function of the change in the magnitude of the solubility parameter of the ethanol/water mixtures (overall polarity, δh = 26.2-48 MPa1/2) is shown. The maximum solubility (16.6 mg/mL) is represented at an inflection point at δh = 31 MPa1/2 (78% ethanol). The δh at the inflection point is very similar to that previously calculated for nevirapine (27.2).23 This suggests that in ethanol/water
mixtures, nevirapine preferentially interacts with ethanol because the solubility shows a maximum at high ethanol concentration. Previous studies have shown that ethanol and water are highly ordered solvents self-associated through hydrogen bonding and, for a drug such as nevirapine (δh > 25 MPa1/2), an optimal cosolvent ratio can be expected between 60 and 80% ethanol in water (δh = 31-35 MPa1/2).24-27 However, the solubility profile for nevirapine in the ethanol/ water mixtures also suggests that two dominant mechanisms dependent on solvent composition are related to the cosolvent action. First, solubility enhancement, although not large, is most probably entropy driven at high water ratios due to an entropy gain caused by a loss of water structure around the hydrophobic moieties of the drug molecule (entropy as the driving force up to 25% m/v ethanol). Up to this ethanol concentration, anhydrous nevirapine was recovered from saturated solutions that were allowed to crystallize. From 25 to 70% (m/v), only the hemihydrate form was recovered. As the solution becomes more ethanol rich, this mechanism probably changes and the solubility is favored by a decrease in enthalpy. However, above 70% (m/v) ethanol, the solubility starts to decrease;possibly related to an unfavorable entropy change;and the dominant mechanism shifts from enthalpy again to entropy. This change coincides with the gradual transition of the hemihydrate to the hemiethanolate and also the solubility maximum. Water activity (aw) is another variable which should be considered when dealing with a solvent system that includes water, especially since ethanol reduces the water activity of aqueous phases and this can change the solubility of a drug and determine the crystal form that precipitates from different water/ethanol mixtures.28 Figure 13 shows the solubility of nevirapine as a function of water activity at 25 C. Values of aw in mixtures of ethanol and water were calculated from the known values29,30 of activity coefficients (γw) and the mole fraction of water (mw) in these mixtures: aw ¼ γw mw Values of aw are fitted to a polynomial in mw, which enables γw to be estimated at any value of mw in the organic solvent þ water mixtures employed for the crystallization of a drug. However, the aw value in the solvent mixtures will be modified by the presence of the dissolved drug because mw will decrease with an increase in the solubility of the drug. The change in mw
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Figure 13. Solubility curve of nevirapine as a function of water activity at 25 C.
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different morphologies. It was seen that these crystal form and shape changes were closely associated with the change in solubility of nevirapine in the solvent mixtures. In turn, the change in solubility was shown to be a function of the change in magnitude of the solubility parameter and changes in water activity. In particular, when ethanol predominates in the solvent medium (>70% m/v), solubility starts to decrease and this change coincides with the solubility maximum and the gradual transition of the hemihydrate to a crystallographically distinct hemiethanolate with a new crystallite morphology. Solute-solvent interactions and differences in the degree of self-association also contributed to the shape of the nevirapine solubility profile in ethanol/water mixtures. Specifically it was shown that there is a correlation between the reduction in water activity, when increasing amounts of water are added to a constant volume of ethanol, and the solubility of nevirapine in ethanol/water mixtures. Acknowledgment. We are grateful to the University of Cape Town, the University of Wisconsin, North-West University, and the National Research Foundation of South Africa (NRF) for research support.
References
Figure 14. Correlation between the reduction in water activity, caused by the solute, and the solubility (at 25 C) of nevirapine when increasing amounts of water are added to a constant volume of ethanol.
can be calculated using the equation for a three-component system proposed by Sachetti,31 and this can then be used to calculate the change in water activity (Δaw): mw ¼
Ωw =Mw Ωw =Mw þ Ωo =Mo þ Sd =1000Fs Md
where Ω is weight fraction, M is molecular weight, Sd is drug solubility (mg/mL), and Fs is the density of the solution (g/mL). The subscripts w, o, and d represent water, the organic solvent, and the drug substance, respectively. In this study, the change in solubility of nevirapine mirrored the change in water activity (Figure 14). Water activity, rather than concentration in the aqueous solution, should therefore be a critical parameter in designing a suitable crystallization process for nevirapine. Conclusion In an effort to determine the best possible ethanol/water mixture to prepare a nevirapine solution with maximum solubility and stability against crystallization, it was observed that when nevirapine is recrystallized from water/ethanol mixtures, both solvated and unsolvated species may appear at various stages, with different crystalline phases displaying
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