CRYSTAL GROWTH & DESIGN
Crystallization and Crystallinity of Fluticasone Propionate
2008 VOL. 8, NO. 8 2753–2764
Darragh Murnane, Christopher Marriott, and Gary P. Martin* King’s College London, Drug DeliVery Research Group, Pharmaceutical Sciences DiVision, 150 Stamford Street, London SE1 9NH, United Kingdom ReceiVed October 1, 2007; ReVised Manuscript ReceiVed January 31, 2008
ABSTRACT: Solubilization of fluticasone propionate (FP) was effected using aqueous solutions of (i) different grades of poly(ethylene glycol) (PEG), (ii) methanol, and (iii) acetone to enable antisolvent crystallization by the addition of water. The solubility of FP in acetone was significantly higher than in PEG 400 or PEG 6000, and FP solubility was observed to be nonideal in either cosolvent. Crystallization of FP was instantaneous upon addition of water as antisolvent, with nucleation occurring during the mixing phase. The smallest crystals were produced in all cases from PEG solvents, which was attributed to a greater degree of nucleation and microcrystals of a size-range were produced. Crystals produced from PEG solvents also displayed a resistance to agglomeration and Ostwald ripening, which was observed to affect the morphology of FP crystallized from either methanol or acetone by the addition of water. In spite of the very rapid kinetics of solid formation, FP crystallized as the stable Form I polymorph from PEG 400 and PEG 6000. Conversely, mechanical milling of highly crystalline particles resulted in the generation of disorder in the crystals, which was apparent from surface dynamic vapor sorption analysis. The generation of FP microcrystals with a small size range from environmentally benign PEG solvents as the stable crystalline form represents an improvement over current crystallization and micronization techniques for the production of inhalable FP. Introduction Fluticasone propionate (FP), an androstane glucocorticosteroid used in the treatment of asthma, is crystallized from organic solvents, whereafter the conventionally crystallized FP particles are processed by jet-mill micronization. This ensures that the particle size is suitable for deposition in the lower airways after inspiration of a formulated product. Micronization produces irregularly shaped particles1 and decreases the crystallinity of FP as a result of generating surface amorphicity.2 The consequence of these latter phenomena are the resultant induced aggregation of FP,3 which in turn can lead to low in vitro respirable fractions when presented as a powder, propellant suspension, or blended with lactose.1 A number of particle engineering approaches have been investigated to improve the aerosolization of drug particles including spray-drying,1 antisolvent micronization,2 and supercritical antisolvent micronization.4,5 Conventional flammable organic solvents are necessary for the production of microparticles, requiring removal from pharmaceutical products and safety considerations during processing. Alternative production techniques have been reported to result in amorphous character1,3 or metastable polymorphs,4,6 and these can be attributed to the very rapid kinetics of solid formation (crystallization) in all these production methods.7 FP is an extremely hydrophobic molecule of very low8 aqueous solubility ( PEG 400 > acetone. Interestingly the PEG 400 and acetone plots intersected between 30-40 mol % cosolvent, above which FP was more soluble in acetone-water than PEG 400-water. Although PEG displays a high solubilization capacity for FP (e.g., over 4 log cycle increases for PEG 400), the absolute solubility is lower in 100% PEG (7.15 ( 0.39 mg g-1 for PEG 400) than in 100% acetone (58.87 ( 0.77 mg g-1). It would appear that a bilinear model17 was more appropriate to describe the solubilization of FP by PEG and acetone expressed as a % w/w. The bilinear model can be explained by the nonideality of PEG and acetone solutions in water arising from extensive intermolecular H-bonding.18,19 Solvent properties vary between the water-rich and cosolvent-rich solutions as a result of specific and nonspecific solvation interactions.20,21 The more effective solubilization by PEG than acetone at low mole fractions was understood on the basis that in breaking down the structure of water, each individual ethylene oxide unit behaves as a small molecular weight solute in its interactions with water.18 Thus the ability to break down the structure of water increases with PEG molecular weight.22 In the cosolventrich region, above the PEG 400 and acetone intersection, solvation is dominated by the properties of the cosolvent. One reason for the higher solubility in acetone than PEG may be that PEG 40023 has a greater specific H-bonding and lower polar component to its partial solubility parameters than acetone.24 Characterization of Crystallization Media: Rheology. Plots of viscosity against concentration of PEG 6000, PEG 400, and FP-PEG 400 crystallization media in water were identical to those previously reported for PEG media15 and are therefore not reproduced here. The regression equations of the above plots are, however, reproduced in Table 2. The viscosity of PEG during antisolvent crystallization is likely to contribute to a mesomixing step25 which can promote localized supersaturation and a resultant higher nucleation rate to ensure a low particle size. Below a concentration of 80% w/w PEG, cloudiness was seen to occur in the FP-PEG 400 media, which was attributable to the precipitation of FP. This was concomitant with a change from the Newtonian behavior of the solutions containing g90% w/w PEG 400 to plastic flow at lower PEG concentrations. Accordingly, a linear plot of shear stress against shear strain possessed a yield stress intercept. There was an increase in the yield stress values as the concentration of PEG in the medium decreased (see Table 3), and the phase volume of FP crystals in the medium increased. Crystallization of Fluticasone Propionate. Effect of Solvent. The CSD of FP particles following antisolvent crystallization from PEG 400 (FP 1) at a supersaturation of ∼7.8 and from methanol (FP 2) are presented in Table 4 (the solubility in methanol-water could not be determined as it was below the limit of detection of the HPLC method). Both solvents could successfully be employed to generate microcrystals of FP. However, the crystals harvested upon termination of the
Table 3. Yield Stress of FP/PEG 400 Crystallization Media at Varying PEG Weight Concentrations in Water concentration of PEG (% w/w)
yield stress (Pa)
10.1 20.2 30.2 40.3 49.7 60.8 70.4 80.5
0.583 ( 0.052 0.488 ( 0.011 0.356 ( 0.005 0.267 ( 0.014 0.209 ( 0.003 0.096 ( 0.010 0.061 ( 0.001 0.044 ( 0.010
crystallization at 30 min after addition of water to the FP/PEG 400 solution were significantly smaller than those produced from methanol (Student’s t test, p < 0.001). In particular the particles displayed a tighter distribution, as shown by the smaller cumulative 90% undersize diameter. This was important because for pulmonary aerosol therapy particles should be in the size range 1-6 µm.26 Previous reports into production of FP microparticles have utilized acetone as the API solvent. It was, therefore, appropriate to compare PEG 400 and acetone as crystallization solvents. The rate of addition of the aqueous antisolvent to solutions of fluticasone propionate in PEG 400 and acetone was demonstrated to affect the particle size distributions (Table 4). Crystals produced from solutions of either PEG 400 (FP 4) or acetone (FP 6) were significantly larger when the antisolvent was added at the slower rate (Student’s t test, p < 0.003). When the antisolvent was added rapidly, despite the higher supersaturation of the acetone crystallization media (FP 5), the particle size was still significantly greater for crystals produced from acetone than from PEG (FP 3) (p < 0.013). When the antisolvent was added slowly, the PEG-derived crystals were again significantly smaller than their acetone counter-parts (p < 0.001). This demonstrated an ability of PEG 400 as a crystallization medium to offer more control over the crystal size compared to acetone. The size of particles produced by the addition of water as antisolvent to PEG solutions of FP was markedly smaller than those produced by precipitation from methanol or acetone solutions, when similar conditions of crystallization were employed. The supersaturation in all systems was several thousand times the equilibrium solubility in the terminal cosolvent mixture. Previous studies on the precipitation of FP from conventional solvents demonstrated extensive crystal growth or agglomeration-based growth of FP,2 which necessitated the inclusion of HPMC or polyvinyl alcohol polymers to produce microcrystals. Crystallization of FP in a precipitation process from PEG 400 appears to overcome the crystal growth common of other solutions at high supersaturations. Crystals produced from PEG 400 demonstrated a higher proportion of smaller particles (demonstrated by the lower D(v,0.1)) than the organic solvents. This suggests extensive nucleation in the case of PEG 400 drug solutions, even in the case where the maximal supersaturation of FP was lower than in acetone media (FP 3 and 4: σPEG ) 8.0 and FP 5 and 6: σacetone ) 10.1). Nucleation should be more rapid in acetone: the higher solubility of FP in acetone predicts a lower interfacial tension10 enabling the formation of a crystal phase during nucleation. The latter observations suggest a difference between the process/rate of generation of supersaturation27 in PEG and conventional solvent media. In antisolvent crystallization, supersaturation is affected by the hydrodynamics of mixing, which in turn depend on the rheological properties of the solvents. The increase in median particle diameter regardless of the solvent (acetone or PEG 400) upon decreasing the antisolvent
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Table 4. Particle Size Distribution (mean ( SD, n ) 5) of Fluticasone Propionate Microparticles
a
experiment
solvent
D(v, 0.1) (µm)
FP 1 FP 2 FP 3 FP 4 FP 5 FP 6 FP 7 FP-ace FP 8 FP 9 FP 10 micronized FP FP acetone kinetic FP PEG 400 kinetic
PEG 400 methanol PEG 400 PEG 400 acetone acetone PEG 400 acetone-water PEG 400 PEG 6000 PEG 400 n/a acetone PEG 400
0.62 ( 0.02 1.90 ( 0.35 0.86 ( 0.02 1.98 ( 0.10 2.99 ( 0.34 4.09 ( 0.16 0.93 ( 0.04 2.73 ( 0.01 0.85 ( 0.02 0.92 ( 0.05 1.94 ( 0.03 1.01 ( 0.02 4.01 ( 0.14 2.17 ( 0.14
The D(v,
0.1),
D(v,
0.5),
D(v,
0.9)
D(v, 0.5) (µm) 2.25 ( 0.09 5.73 ( 0.35 2.90 ( 0.13 6.21 ( 0.54 7.80 ( 1.10 15.11 ( 0.76 2.96 ( 0.14 13.60 ( 0.04 2.50 ( 0.10 3.21 ( 0.19 6.14 ( 0.17 3.06 ( 0.05 14.81 ( 0.74 6.84 (0.10
a
D(v, 0.9) (µm) 7.59 ( 0.24 20.85 ( 1.83 8.80 ( 0.52 16.54 (1.49 15.31 ( 2.36 32.69 ( 2.02 8.02 ( 0.33 28.40 (0.08 6.73 ( 0.19 9.13 ( 0.38 17.98 ( 0.96 7.20 ( 0.15 34.22 ( 1.41 18.84 ( 0.29
σmax 7.8 8.0 8.0 10.1 10.1 8.0 7.9 8.0 8.1 n/a 8.3 7.8
represent the 10%, median and 90% cumulative volume undersize diameters, respectively.
Figure 2. Concentration of fluticasone propionate (FP) in the crystallization medium as a function of time during the addition of water as antisolvent to PEG 400 solutions (A) and acetone solutions (B). (2) and (9) represent the ideal solubility curve and actual concentrations of FP determined in PEG 400 media, respectively (mean ( SD, n ) 4); (b) and (∆) represent the ideal and actual concentrations of FP in acetone media, respectively (mean ( SD, n ) 2).
addition rate supports the importance of the rate of generation of supersaturation. A slower addition rate of antisolvent promotes more homogeneous dispersion throughout the medium in the crystallizer favoring the generation of homogeneously dispersed supersaturation.28 The importance of a higher extent of nucleation in the PEG systems relative to acetone was demonstrated by the significantly higher increase in the D(v,0.1) particle size value of the former (Table 4), upon decreasing the antisolvent addition rate, although the relative increase in the median particle diameter (a doubling) was similar for both solvents. The smaller size of FP crystallized from PEG under similar experimental conditions may arise from the slower growth rate of crystals in PEG than in aqueous-organic solvents, although reported growth rates in PEG media are high.29 Although growth rates may be high in PEG solvents of high viscosity, solvent viscosity does affect the mixing of crystallization solvent promoting segregation of mixing into the mesoscale.25 The resultant localization of supersaturation can ensure a high nucleation rate,30 whereas crystal growth depends more on mean crystallizer supersaturation.31 The localized creation and dissipation of supersaturation and the subsequent extensive nucleation of a large number of small particles ensures the mean
supersaturation necessary for further growth of these crystals is no longer available. The low crystal growth in PEG solvents is also likely to arise from the fact that supersaturation exists (see Figure 2 and the following section for discussion) when viscosity was at its highest (e.g., range 55.3-17.6 mPa · s corresponding to 85-60% w/w PEG 400 in water, for FP/PEG 400 crystallization media) limiting the diffusion of molecules and nuclei. Supersaturation Kinetics. By using a high-precision infusion pump, it was possible to control the rate of addition of the aqueous antisolvent to solutions of FP in PEG 400 and acetone. Samples (0.2-1.2 g) removed by syringe from the crystallization liquor (at 30 s, 60 s, and then at 60 s intervals up to 9 min) were filtered, diluted appropriately, and assayed for FP content by HPLC. From the knowledge of the weight of water added, it was possible to calculate the concentration of PEG in the crystallization medium; and from the solubilization plots it was possible to construct an ideal curve of solubility of FP as a function of time (and therefore, as a function of added water). The concentration of drug in the crystallization medium was determined by HPLC and the actual mean supersaturation was calculated. These plots are presented in Figure 2. Note that unlike Figure 1, where FP solubility was expressed on a
2758 Crystal Growth & Design, Vol. 8, No. 8, 2008
Figure 3. Plot of the supersaturation of fluticasone propionate as a function of time during the addition of water as antisolvent to PEG 400 (2) and acetone (9) solutions (mean ( SD, n ) 4 for PEG 400 and n ) 2 for acetone).
logarithmic scale, concentrations are expressed on a linear scale in Figure 2. Particle formation occurs simultaneously with mixing in highly supersaturated systems.32 It is a challenge to calculate local supersaturation and only mean supersaturation throughout the medium can be measured.31 However, the above approach allowed an estimation of the process of generation of supersaturation during the mixing phases. Crystallization was performed using a similar level of theoretical supersaturation (σmax ) 8.3 for acetone and σ ) 7.8 for PEG 400). Because of error in determination of the solubility of FP in the acetone/water crystallization media (solubility at 12.5% w/w acetone in water: 1.18 ( 0.45 µg g-1, i.e., % RSD ) 37%), the resultant determination of σmax for FP in the acetone/water mixtures according to eq 1 had wide confidence intervals. As such, no significant difference resulted between the σmax for the acetone and PEG 400 crystallization batches. The plots of concentration as a function of time during the initial addition of water demonstrate that there is a greater window of supersaturation in the PEG 400 crystallization medium than in the acetone system. The only point at which a statistically significant supersaturation was shown in the acetone plot was at 4 min (p ) 0.038). The confidence limits for this determination were large as a result of the plot being based on only two repeat determinations of concentrations. For the PEG 400 medium, at all time points investigated, the actual concentration in the medium was significantly different to the theoretical solubility (p < 0.027). Although the maximal supersaturation drive for the crystallization process (σmax (eq 1) calculated for the final solvent composition) for crystallization was identical, a plateau was identified in the acetone system, where the determined concentration of FP in the medium remained constant over the first 4 min. A plot of the actual supersaturation as a function of time is presented in Figure 3, which demonstrates clearly the higher supersaturation present in the PEG 400 system, over the time course of study. The plots of supersaturation were significantly different at 0, 0.5, 2, and 4 min (p < 0.02). The other data points did not show significant differences due to the wide confidence intervals for the acetone values. The above findings demonstrated the difficulty in using theoretical values such as σmax based on instantaneous mixing prior to nucleation, where actual supersaturation depends on the shape of the solubilization
Murnane et al.
curve and can vary during the mixing period. Crystals produced from acetone in this kinetically monitored study were shown to be significantly larger (p < 0.003) than those from PEG 400 (Table 4). It was only possible to study the desupersaturation over the initial stages of antisolvent addition (9 min, representing ∼12% of the total water volume addition). Comparison of the two supersaturation plots demonstrated that the supersaturation of the PEG 400 batches was dissipated by peaking to higher levels than acetone batches during the initial phases of antisolvent addition. The lower supersaturation peaks in acetone-water systems cannot solely be ascribed to the fact that the starting concentration of the FP-PEG solutions was closer to saturation solubility than the FP-acetone solutions. Small particle sizes require high rates of nucleation resulting from the peaking of supersaturation. Even when batches of FP were crystallized from acetone at concentrations closer to equilibrium solubility (e.g., FP 5), their particle sizes were still larger than PEG-crystallized FP (e.g., FP 3). In Situ Analysis. The crystallization process of FP from PEG 400 (FP 7) was monitored using FBRM and PVM. The reverse addition procedure was necessary to accommodate the in situ probes. It was observed by FBRM that a rapid precipitation event occurred, with an increase in the counts of crystals with chord lengths in the size range of 1-10 µm (Figure 4). The trend graph also shows that crystallization was essentially complete about 75 s after addition of the drug solution to water. There was also a rapid rise in particles in the range 10-102 µm, whereas counts of larger sized particles remained low, and showed no increase over time. There were no significant changes in the count statistics for any size interval over the time course of the crystallization (i.e., 13 min). The particles demonstrated a median chord length of 6.35 µm at the termination of crystallization, and a square-weighted mean chord length of 46.92 µm. The difference between the chord length and median diameter from laser diffraction (Table 4) is most likely a result of the acicular particle shape. Reduction of the stirrer speed to 100 rpm resulted in a decrease in the count of fine particles and a rise in the counts of particles with chord lengths in the range of 102-500 µm (Figure 4). Upon increasing the stirrer speed to 800 rpm the mean particle size and the weighted chord length distribution was seen to increase (Figure 4). At 1200 rpm the mean particle size and the distribution had shifted to lower sizes than when the medium was stirred at 100 rpm. Representative PVM images show that at 100 rpm (Figure 5) the crystal population appeared as large flocs of loosely flocculated small needles which were mobilized at 800 rpm but ruptured to form a population of individual and small needle-shaped crystals upon increasing the shear rate corresponding to that generated by a stirrer speed of 1200 rpm. The presence of flocs could account for the presence of a yield point in the rheograms. Crystal Morphology. Observations from SEM analysis suggest further explanations for the control of crystal size distribution by PEG when used as the solvent for aqueous precipitation (Figure 6). At high magnification it was clear that the plate-like crystals produced from acetone and methanol consisted of needles of much smaller diameter which had consolidated by cementation of particles. This situation did not occur following PEG crystallization, leading to the different macromorphology33 of the crystals. The cohesion of particles of FP crystallized from PEG observed in Figure 6C,D is a feature typical of micron-sized particles in the dry state rather than agglomeration/cementation discussed above. The final
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Crystal Growth & Design, Vol. 8, No. 8, 2008 2759
Figure 4. (A) Trend plot demonstrating the size of the population (counts s-1) in the indicated ranges of chord length as a function or time measured by focused beam reflectance measurement (stirrer speed 500 rpm); and (B) the weighted chord length distribution (% total counts s-1) as a function of chord length at the indicated stirrer speeds (right) for fluticasone propionate crystallized from PEG 400.
Figure 5. Videomicrograph of fluticasone propionate crystallized from PEG 400, during the late stages of the crystallization experiment at a stirrer speed of 100 rpm (A) and at 1200 rpm (B).
macromorphology of FP crystallized from PEG was acicular, while that of acetone- and methanol-crystallized FP was platelets of consolidated smaller needles. As such, crystals of FP produced by antisolvent crystallization demonstrated a tendency to a needle-shaped habit. The macromorphology of a crystal depends on the relative growth rates of the different faces of the crystal and may be altered by solvent choice as well as supersaturation levels. The growth rates of different crystals faces are affected to varying extents by an increase in supersaturation.34 The latter observation may explain the tendency for needle-like crystals (prior to agglomeration) regardless of the solvent and was previously proposed to explain paracetamol morphology.35 The presence of high generalized, as opposed to localized, supersaturation persisting after nucleation can provide the opportunity for crystal growth by agglomeration and cementation arising from the presence of well macromixed homogeneous environment. This situation is a common means by which crystal growth occurs in highly supersaturated precipitation crystallization.32,36 Several reasons can explain why the cementation of FP crystals produced from acetone and methanol did not occur in PEG systems. (1) Desupersaturation proceeded early during antisolvent addition that the supersaturation necessary to allow crystal growth during cementation was no longer available. (2) A different rate of generation of supersaturation existed due to
the different solubilization curves. (3) PEG molecules can adsorb to the crystal interfaces and prevent agglomeration occurring. It is difficult to conclude which of these reasons is truly operational. PEG is known to adsorb onto hydrophobic solid surface;37 however, PEG was not shown to act as a crystal growth inhibitor in previous investigations.2,13 Because of polymer chains’ resistance to flow, PEG may accumulate at hydrophobic surfaces38 and it is attractive to conclude that crystalline bridge formation is prevented by steric hindrance. An alternative explanation relies on nucleation from PEG systems occurring during the phase when PEG concentration, and hence solution viscosity was at its highest. High viscosity can prevent collision of the particles and when combined with a lower growth arising from the low mean supersaturation, cementation (bridge formation) between colliding particles is avoided. The smaller particle size of FP when crystallized from PEG arose from the rapid nucleation at early stages of crystallization combined with decreased crystal aggregation and cementation. Solid-State Characterization of FP Microparticles. FP is routinely crystallized from organic solvents of middle polarity including ethyl acetate for primary crystallization and from acetone for purification/recrystallization.39 Crystallization from such solvents results in the Form I polymorph of FP,40 while a second (metastable) polymorph (Form II) has been reported for
2760 Crystal Growth & Design, Vol. 8, No. 8, 2008
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Figure 6. Scanning electron micrographs of fluticasone propionate particles: (A) crystallized from methanol-water (FP 3; × 10000); (B) crystallized from acetone-water (FP 5; × 5000); (C) crystallized from PEG 400-water (FP 8; × 10000); and (D) crystallized from PEG 6000-water (FP 9; × 10000).
FP crystallized from acetone or ethanol using supercritical antisolvent precipitation.6 The monotropic polymorphism arises due to a different packing of the monomers in the crystal lattice. In the Form I polymorph molecules are H-bonded in zigzag chains where adjacent chains are stacked parallel, while in Form II, these chains are stacked in an antiparallel geometry.6,41 Representative diffractograms of FP crystals and microparticles are presented in Figure 7. The positions of the peaks are identical in each diffractogram, demonstrating that the same polymorph of FP was produced irrespective of the solvent system employed. There were, however, some differences in the intensity of the peaks at several diffraction angles. Differences in the crystallinity are generally indicated by differences in the peak intensities of several peaks in the PXRD pattern. However, the apparently lower crystallinity of PEG-crystallized and micronized FP than FP crystallized by slow cooling may be a result of preferred orientation and the lower particle sizes of the former particles. The presence of texture in powder specimens systematically distorts the intensity ratios of peaks,42 and needle-shaped crystals (such as amphibole asbestos) have been specifically highlighted as causing preferred orientation effects.43 Thermal analysis by differential scanning calorimetry, however, did not reveal the presence of a glass transition1 (characteristic of amorphous disorder) or a polymorphic conversion endotherm5 in any of the microparticles (data not shown). As the fusion of FP crystals is concomitant with degradation, it
was not possible to determine enthalpies of fusion to examine changes in crystallinity. Importantly, the FP crystals produced by antisolvent micronization from PEG 400 (FP 8) and PEG 6000 (FP 9) displayed crystallinity which was no poorer than that of the commercially available micronized material. Indeed, similar peak intensities at certain diffraction angles were observed for FP crystallized by cooling of an acetone-water solution (FP-ace) and the two microparticle batches. FP-ace and commercial mFP both showed a doublet at 2θ angle of 16.0° and 16.4° with the highest intensity peak being at 16.0°. This situation was reversed in the case of all crystals produced using PEG as the solvent. While this might be related to a decreased crystallinity of the samples, the different relative intensities of the doublet may be related more to the different crystal habit observed in FP crystallized from PEG. Samples of FP crystallized from PEG 400 and of FP recrystallized from acetone-water (FP-ace) were subjected to ball milling. A haloing of the baseline was seen with PEG-FP (Figure 8) when it was subjected to the milling forces, indicative of increased disorder in the crystallinity of the powder. This was also shown by a decrease in the intensity of the diffraction peaks. The complete merging of the peaks at 16.0° and 16.4° was evident. FP-ace crystals also demonstrated a decrease in crystallinity upon mechanical processing (Figure 9). This was represented by a broadening of the peaks and a decrease in
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Crystal Growth & Design, Vol. 8, No. 8, 2008 2761
Figure 7. Representative powder X-ray diffractogram of fluticasone propionate (FP) particles: (A) commercial micronized FP; (B) FP 8 crystallized from PEG 400; (C) FP 9 crystallized from PEG 6000; and (D) FP crystallized from acetone-water by cooling.
Figure 8. Powder X-ray diffractograms of fluticasone propionate particles crystallized from PEG 400 (FP 10) after (A) and before (B) mechanical processing.
intensity. Although the effects of particle size and preferred orientation cannot be discounted, the observation of decreased crystallinity of FP-ace particles following milling is in accordance with previous reports of the deleterious effects of milling on FP.2 Dynamic Vapor Sorption Analyses. No obvious signs of recrystallization events were observed in any of the samples investigated (Figure 10). An identical sorption profile was observed for the first and second sorption cycle when FP 10 was analyzed, with a final weight gain of 0.45% at the end of each cycle. A similar finding was obtained for micronized FP (mFP), with a final uptake of 0.25%. It was not possible to remove all water vapor during the second drying period at 0% relative humidity, and there was a change in slope of the sorption profile near the end of the initial high relative humidity phase for mFP, which may be due to bulk absorption. FP-ace demonstrated only a small uptake of water. The greatest sorption
Figure 9. Powder X-ray diffractograms of fluticasone propionate crystallized from acetone-water (FP-ace) after (C) and before (D) mechanical processing.
was observed for the milled FP-ace material with a lower water uptake (0.56%) on the second sorption cycle than on the first cycle (0.64%). DVS can identify the presence of amorphous material on the surface of crystalline material,44 by detecting the crystallization and relaxation of disordered regions as a function of relative humidity.45 No recrystallization event was observed in any of the DVS traces in this study, presumably due to the hydrophobicity of FP.46 The absence of a recrystallization event does not signify the absence of amorphous content.44 The higher specific surface area of microparticles (FP 10, mFP, and milled FP-ace) compared to FP-ace explains the higher mass of vapor sorption which was observed, particularly when the total amorphous content as determined by PXRD and DSC is low.46 Mechanical processing of FP-ace material resulted in a greater uptake on the first sorption cycle than the second. This was
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Figure 10. Dynamic vapour sorption profile of (A) micronized fluticasone propionate particles and FP particles produced by antisolvent crystallization from (B) PEG 400 (FP 10), cooling crystallization from acetone (FP-ace) (C) before and (D) after milling. The plots represent the % change in mass (left ordinate and solid line) as a function of time (abscissa) when the relative humidity changes as a function of time (right ordinate and dotted line).
consistent with moisture being sorbed into the disordered regions as well as adsorbed onto the crystal surface on the first cycle, and only onto crystalline surfaces on further cycles.44 Crystallinity of FP Microcrystals. It was anticipitated that FP crystals produced by antisolvent crystallization from PEG would contain amorphous content, disordered regions or be present as the metastable polymorph. Metastable crystal forms or highly flawed crystals (containing defects and disrupted packing) are typically produced by rapid crystallization processes,7 and precipitation processes frequently lead to the generation of amorphous particles,31,32 including FP. Indeed the production of metastable forms has been reported from antisolvent crystallization of FP,3,4 flunisolide,47 and amorphous forms of budesonide48 and beclometasone dipropionate.13 FP crystallization from PEG occurred in a rapid time frame caused by the peaking of very high supersaturation upon addition of water. Despite this rapid crystallization, the stable polymorph (FP Form I) resulted, and no evidence of crystalline metastability was displayed. It cannot be assumed that the most stable crystal form will be produced in antisolvent crystallization. According to the Ostwald rule of stages, the initial crystalline form will be one which occupies the energy minimum closest to the solution state (i.e., metastable form),49 and the crystal may be kinetically trapped in this form.50 However, nucleation is a dynamic process depending on kinetic as well as thermodynamic considerations.51 The balance between thermodynamic and kinetic control of nucleus formation52 can ensure the stable solid-state form in a monotropic system preferentially crystallizing at all supersaturation levels as a result of the activation energy for nucleation and the rate of generation of supersaturation for the respective dimorphs. Primary nucleation stages are interspersed with polymorphic transformations involving dissolution of a metastable phase and growth of a stable polymorph.53 This should, however, be limited by the rapid rate of crystallization in
the current FP-PEG system (see kinetic analysis above). In situ analysis of the crystallization process by PVM did not unveil any alteration in crystal morphology during the time course of crystallization; thus any metastable form of FP can only have been formed and replaced during the nucleation period. Nucleation may occur through a metastable liquid crystal phase,49 regional high density fluctuations,51 or an amorphous phase32,54 rather than a distinct metastable crystalline polymorph. It is possible that such metastable nuclei precursors crystallize as either the stable or metastable polymorph. Because of the rapid nucleation and crystallization at high supersaturation in the PEG systems such crystallization from a metastable nucleus represents a possible route for the precipitation of the stable polymorph. It was reported that upon crystallizing substances from aqueous-PEG solvents that segregation of antisolvent and drug solutions can be achieved depending on the stirring conditions,55 and this can result in delayed induction times for nucleation. The authors subsequently described the production of stable polymorphs due to delayed nucleation.56 Conversely metastable and mixtures of crystal forms were produced when rapid homogeneous mixing was employed. The stable crystal form was isolated for FP in the current work, even though mixing conditions were such that segregated quasi-emulsion droplets could never form and rapid nucleation occurred. Wang et al.55 employed 60% w/w aqueous PEG solutions of drugs exhibiting significantly lower viscosity than the 100% PEG employed in the current study, in which FP was solvated. PEG is completely hydrated in a 60% w/w aqueous solution, thus obviating the requirement for FP molecules to be desolvated from the polymer and for PEG to be hydrated for reduction of FP solubility. The hydration step in combination with slower diffusivities of water and FP molecules in the viscous PEG medium may be responsible for crystallization of the stable FP polymorph upon initial antisolvent addition according to the kinetic-thermodynamic balance. Further antisolvent addition
Crystallization of Fluticasone Propionate
leads to a competition between growth of the initially produced nuclei, further nucleation, and dilution. Any metastable nuclei produced are more likely to dissolve upon dilution due to their higher solubility, while the density of microparticles present in the crystallizer promotes heterogeneous nucleation. Stable FP crystals formed initially can therefore act as seeds to direct further nucleation of that stable form I polymorph. Conclusions It was possible to crystallize fluticasone propionate with a controlled particle size range and a tight particle size distribution by antisolvent crystallization from PEG solutions by the addition of water. Agglomeration leading to a broad particle size distribution (and particles with large median diameters) was not observed. The use of PEG as a crystallization solvent seemed to obviate agglomeration effects, which were observed when FP was crystallized at a similar level of maximal supersaturation from two conventional organic crystallizing solvents: methanol and acetone. The formation of microcrystals was associated with rapid desupersaturation kinetics, and this resulted in the dissipation of the majority of supersaturation when the PEG solution was at its most viscous and when localized supersaturation was most likely to occur. Further work is necessary to control accurately the mixing conditions (e.g., employing a jet reactor) in order to characterize the process for the generation of supersaturation further. Crystallized FP microparticles were isomorphic crystals of FP, present as the Form I polymorph. The crystals formed were acicular, which was also noted to be the fundamental morphology of crystals grown from organic solvents. FP microcrystals demonstrated an equivalent size distribution to the commercial micronized material, and advantageously demonstrated a high crystallinity, with the absence of amorphous material or metastable polymorphs. The advantage of crystallizing FP by antisolvent micronization for use in inhalation therapy was shown by the improved crystallinity of the microcrystals compared to that of milled FP. The production of microcrystals of FP demonstrated the suitability of amphiphilic crystallization for further APIs, even at very high supersaturation levels. It also demonstrated the opportunity to control micromeritic properties such as shape and size distribution and the crystalline form of particles crystallized from PEG solvents. Acknowledgment. The authors acknowledge King’s College London and MedPharm Ltd for financial support. We thank Mr. Ian Haley and Mettler-Toledo AutoChem for the provision of the Lasentec FBRM and PVM analysis units and are very grateful for Mr. Haley’s advice and expertise, which contributed to this study.
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CG700954T
