Miscibility of Itraconazole–Hydroxypropyl Methylcellulose Blends

Nov 15, 2015 - Miscibility of Itraconazole–Hydroxypropyl Methylcellulose Blends: Insights with High Resolution Analytical Methodologies. Hitesh S. P...
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Miscibility of Itraconazole-Hydroxypropyl Methylcellulose Blends- Insights with High Resolution Analytical Methodologies HItesh S. Purohit, and Lynne S. Taylor Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00761 • Publication Date (Web): 15 Nov 2015 Downloaded from http://pubs.acs.org on November 21, 2015

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Molecular Pharmaceutics

Miscibility of Itraconazole-Hydroxypropyl Methylcellulose Blends- Insights with High Resolution Analytical Methodologies Hitesh S. Purohit† and Lynne S. Taylor*,† †

Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States

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ABSTRACT Drug-polymer miscibility is considered to be a prerequisite to achieve an optimally performing amorphous solid dispersion (ASD). Unfortunately, it can be challenging to evaluate drugpolymer miscibility experimentally. The aim of this study was to investigate the miscibility of ASDs of itraconazole (ITZ) and hydroxypropyl methylcellulose (HPMC) using a variety of analytical approaches. The phase behavior of ITZ-HPMC films prepared by solvent evaporation was studied before and after heating. Conventional methodology for miscibility determination i.e. differential scanning calorimetry (DSC) was used in conjunction with emerging analytical techniques; fluorescence spectroscopy, fluorescence imaging and atomic force microscopy coupled with nanoscale infrared spectroscopy and nanothermal analysis (AFM-nanoIR-nanoTA). DSC results showed a single glass transition event for systems with 10% to 50% drug loading, suggesting that the ASDs were miscible, whereas phase separation was observed for all the films based on the other techniques. The AFM-coupled techniques indicated that the phase separation occurred at the sub-micron scale. When the films were heated, it was observed that the ASD components underwent mixing. The results provide new insights into the phase behavior of itraconazole-HPMC dispersions and suggest that the emerging analytical techniques discussed herein are promising for the characterization of miscibility and microstructure in drug-polymer systems. The observed differences in the phase behavior in films prepared by solvent evaporation before and after heating also have implications for processing routes and suggest that spray drying/solvent evaporation and hot melt extrusion/melt mixing can result in ASDs with varying extent of miscibility between the drug and the polymer. KEYWORDS: Amorphous solid dispersion, miscibility, fluorescence, atomic force microscopy.

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Molecular Pharmaceutics

INTRODUCTION Administration of a drug in its amorphous form, in particular for poorly water soluble compounds, may be desirable as this form has a higher apparent solubility as compared to the crystalline forms. The solubility advantage of the amorphous form stems from its higher energy, and subsequent dissolution can lead to the formation of a supersaturated solution1 provided that the crystallization of the drug from the solid and from the solution state is inhibited.1-3 Amorphous solid dispersions (ASDs), whereby the drug is dispersed into a polymeric matrix are becoming increasingly prevalent as a solubility enhancing formulation strategy, largely because of the increasing number of low solubility compounds that are being encountered in the drug development pipeline.4, 5 The polymer can function as a crystallization inhibitor of the drug both from the solid formulation and from the supersaturated solution generated following dissolution6 and if the polymer is sufficiently hydrophilic, can also promote wetting and dissolution. It is commonly thought that, in order to produce an optimized ASD formulation, the drug and polymer should be miscible at a molecular level.3, 7 A phase separated system would be expected to have decreased stability as a result of faster crystallization of the drug from the matrix during storage8 and dissolution, resulting in loss of product performance. Therefore, miscibility evaluation is essential to understand ASD properties and performance. A wide variety of analytical techniques such as solid state nuclear magnetic resonance,9-12 local thermal analysis,1316

X-ray diffraction (XRD),17-19 mid-infrared spectroscopy (IR),20,

21

differential scanning

calorimetry (DSC)22-26 and imaging techniques such as atomic force microscopy (AFM),27, 28 scanning electron microscopy (SEM),29 transmission electron microscopy (TEM)30 and confocal Raman imaging31-33 have been utilized to characterize drug-polymer systems in terms of miscibility. A majority of these techniques suffer from limitations. Bulk vibrational

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spectroscopic techniques provide no size information about phase separated domains while imaging spectroscopic methods lack the spatial resolution to characterize very small phase separated domains because the spatial detection limit is restricted by the diffraction limit34 of the light source used. Techniques with high spatial resolution such as AFM, SEM, TEM lack chemical specificity. DSC, which is the most widely used analytical technique for miscibility characterization of drug-polymer blends has its own limitations due to its non-isothermal nature.35, 36 AFM in combination with nanoscale infrared spectroscopy has been used to resolve nanoscale features in organic films,37 study viruses in bacterial cell38 and nanoscale spectroscopy of living cells,39 taking advantage of the high spatial resolution of AFM and the chemical specificity offered by IR analysis. Recently, AFM in combination with nanoscale infrared spectroscopy has been used to evaluate miscibility between two components.40, 41 The goal of the current study was to further evaluate the application of AFM-nanoIR for characterizing drug-polymer miscibility in conjunction with the development of an emerging approach based on fluorescence spectroscopy/imaging. Fluorescence spectroscopy is a widely used analytical technique for the characterization of biological,42 polymeric43-47 and more recently, amorphous pharmaceutical systems.48-50 Herein, fluorescence microscopy and spectroscopy were used as a complement to AFM-nanoIR studies to evaluate the miscibility of a model drug-polymer system, using the environment sensitive probes, pyrene and prodan.51-57 The model drug-polymer system chosen for evaluation consisted of itraconazole and hydroxypropyl methylcellulose; the miscibility behavior of this particular drug-polymer combination at different drug loadings has been debated in literature.58-60 This drug-polymer combination is found in marketed ASD formulations Sporanox® and OnmelTM. By

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Molecular Pharmaceutics

implementation of the aforementioned experimental techniques, the impact of polymer amount and temperature on the miscibility and domain structure of the ITZ-HPMC system was studied. MATERIALS Itraconazole (ITZ) was purchased from Attix Pharmaceuticals (Ontario, Canada), methanol, tetrahydrofuran and dichloromethane were procured from Macron chemicals (NJ, USA). Prodan was obtained from AnaSpec Inc. (CA, USA) while pyrene was obtained from Sigma Aldrich Co. (MO, USA). Hydroxypropylmethylcellulose (HPMC) 606 grade was a gift from Shin-Etsu Chemical Co. (Tokyo, Japan). The structures of the probes, ITZ and HPMC are shown in Figure 1. METHODS Preparation of Drug-Polymer Solutions Solutions containing 50:50, 40:60, 30:70, 20:80 and 10:90 w/w ITZ-HPMC were prepared by dissolving specific amounts of ITZ and HPMC in a 50:50 v/v mixture of methanol (MeOH) and dichloromethane (DCM) in such a way that the solid content in the solution was 20 mg/mL. Two sets of these solutions were prepared. The first set of solutions was used as is, whereas the second set of solutions was further subdivided into two subsets. The fluorescent probe, pyrene, was added to one subset while the fluorescent probe, prodan, was added to the second subset. Addition of the probes was performed by dissolving them in 1:1 v/v mixture of MeOH:DCM and adding the probe stock solutions to the drug-polymer solutions to give a probe concentration of 0.01% w/v (i.e. 100µg/mL). The solutions without the fluorescent probes were used for atomic force microscopy (AFM) and nanoscale infrared (nano-IR) spectroscopy while the solutions containing the probes were used for fluorescence analysis. Solution containing 50:50 ITZ:HPMC

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in 1:1 v/v methanol:tetrahydrofuran (MeOH:THF) were also prepared to investigate if solvent choice has an impact on the phase behavior. Fluorescence Spectroscopy and Imaging Drug-polymer solutions containing the probes were used for fluorescence analysis. A 100µl aliquot taken from each solution was placed onto a quartz coverslip and spun at 3000 rpm for 30s using a KW-4A spin coater (Chemat Technology Inc., CA, USA). A similar procedure was repeated for solutions containing only ITZ and HPMC. The drug only and polymer only solutions served as controls for fluorescence. The resultant thin films were then dried under vacuum for 24h and analyzed using a Shimadzu RF-5301-PC Spectrofluorophotometer (Kyoto, Japan) to obtain the emission spectrum of the fluorescent probe in the films. The films were also imaged using an Olympus BX-51 fluorescence microscope (NY, USA). The filter setting used in the microscope provided excitation from 330- 380nm and emission from 420nm onwards. Fluorescence analysis using the spectrofluorophotometer and the fluorescence microscope was performed before and after heating the films for 2 min at 165°C. Bulk Reference Infrared (IR) Spectra Collection Solutions containing ITZ alone and HPMC alone were prepared in a 50:50 v/v mixture of methanol and dichloromethane. The solutions were then spin coated onto KRS-5 substrates and dried under vacuum. Bulk IR spectra were collected in transmission mode using a Bruker Optics Vertex 70 model IR Spectrophotometer (MA, USA) equipped with a globar infrared source, a KBr beam splitter and a DTGS detector. 64 scans were co-added in the spectral range of 5004000 cm-1. The detector and the sample compartment were continuously flushed with dry air to avoid interference from the moisture present in air. Preparation of Bulk Amorphous Solid Dispersions

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Powdered amorphous solid dispersions (ASDs) of various drug-polymer ratios were prepared by rotary evaporation of the drug-polymer solutions under reduced pressure in a Buchi Rotavapor-R (DE, USA) equipped with a Yamato BM-200 waterbath at 45°C. The dispersions thus obtained were further dried for at least 24h under vacuum prior to use. Differential Scanning Calorimetry Thermal analysis on the samples obtained by rotary evaporation was performed using a TA Q2000 DSC equipped with a refrigerated cooling accessory (RCS) (DE, USA). Approximately 5 mg of the dispersion was placed in a Tzero aluminum pan with a pin hole in the lid and heated at a rate of 2°C/min up to 170°C with modulation of 1°C every 60s. The pan was maintained isothermally at 170°C for 2 min and quench-cooled to 0°C. Reheating of the samples was performed until 170°C with modulation, and changes in the thermal events were monitored. Indium and tin were used to calibrate the temperature scale and enthalpic response. Atomic Force Microscopy (AFM) and Nanoscale Infrared Spectroscopy (nano-IR) Drug-polymer solutions were spin coated onto ZnSe prisms and dried under vacuum. Measurements were performed using a nano-IR™ AFM-IR instrument (Anasys Instruments, Inc., Santa Barbara, CA). This technique overcomes the diffraction limitations of the light that limits spatial resolution in conventional infrared instruments by combining an AFM to a tunable IR laser source thereby providing spatial resolution in the sub-micrometer range. Briefly, the IR radiation from the laser source illuminates the sample through the prism which leads to thermal expansion of the sample at certain frequencies due to absorption of IR radiation. The higher the absorption of radiation of a particular frequency, the higher the thermal expansion. Thermal expansion of the sample increases the amplitude of the cantilever frequency as the AFM cantilever tip is in contact with the surface of the sample. An amplitude vs. wavenumber graph is

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generated by the software by measuring the ringdown amplitudes over a wide range of wavenumbers. Interested readers are referred to Marcott et al.61 for more information on the technique. Data collection on the samples was performed using the cantilever in contact mode. The IR laser was operated at 1mW power and the range of data collection was from 1600 to 1800 cm-1 with co-addition of 256 scans and 4 cm-1 spectral resolution. The scan rate of the cantilever tip was 0.06 Hz and the X and Y resolution was kept 256 points. The contact frequency of the tip was 15KHz. Nanoscale Thermal Analysis (nanoTA) The nanoTA technique utilizes the high spatial resolution of AFM together with a thermal probe to obtain nanoscale thermal data. The nanoTA module along with the nanoTA probe were employed for data acquisition (Anasys Instruments, Santa Barbara, CA). Unlike AFM-nanoIR experiments, AFM images were collected in the tapping mode. The instrument typically gives a plot of deflection versus applied voltage and therefore the voltage has to be converted to a temperature scale by calibration. Temperature calibrations were performed using three crystalline polymers with melting points covering the temperature range of interest. The polymers used were polycaprolactone (melting temperature, Tm=55 ºC), polyethylene (Tm=116 ºC) and polyethylene terephthalate (Tm=235 ºC). Once calibrated, a 40:60 ITZ-HPMC film was tested using this methodology to evaluate the thermal response of the phase separated domains. After acquiring an AFM image using the nanoTA probe, thermal data from the discrete domains and the continuous phase were obtained. The probe was heated at 2 ºC/s and a deflection versus temperature plot was obtained. A decrease or a plateau in the plot represents the softening point which, in present case, corresponds to the transition temperature of the material from where the scan is collected.

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Molecular Pharmaceutics

RESULTS Fluorescence Spectroscopy The probes used in this study are environment sensitive fluorescent probes that show a change in the peak ratio of the emission peaks, I/III, in the case of pyrene62 and a change in emission wavelength maximum in the case of prodan55, 63 as the polarity of the surrounding environment changes. These probes typically show a decrease in the I/III ratio (pyrene) or wavelength (blue shift, prodan) when the environment is less polar and an increase in the I/III ratio or wavelength (red shift) when the environment is relatively more polar. Thus, both pyrene and prodan register a more hydrophilic environment when dispersed in pure HPMC relative to when dispersed in amorphous ITZ (Table 1). The pyrene I/III peak ratio and the maximum emission wavelength of prodan in various ITZ-HPMC films are summarized in Table 1. Before heating, these values for both probes are very close to those observed for the probes in the pure amorphous ITZ films for all the drug-polymer ratios studied. This suggests that the environment sensitive probes are preferentially present in an ITZ rich phase in all the films and thereby register a relatively hydrophobic environment similar to that of pure ITZ. These results are not in accordance with those obtained using DSC (see DSC section of results) as the DSC data suggests the formation of a single-phase miscible system, as inferred from a single Tg, whereas the fluorescence results point toward phase separated systems. A characteristic feature of pyrene fluorescence, in addition to its complex five peak emission spectrum, is the emergence of a broad emission band at approximately 470 nm due to formation of excited state complex called excimers.64 The excimer peak appears when two pyrene molecules that are in close proximity to each other (~5-10 Å) in their ground state interact in their excited state upon absorption of exciting radiation.42, 65 When such a peak is a result of

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excited state interaction of two different molecules, it is referred to as an “exciplex” peak.66 An exciplex peak was obtained in pure ITZ films and in all the drug-polymer ratios studied. Figure 2a shows such a representative exciplex peak in addition to the five peak spectrum of pyrene in a 10:90 ITZ:HPMC ASD film. The appearance of an exciplex peak in pure ITZ films containing pyrene results from the interaction of ITZ with that of excited pyrene molecules. Various examples of pyrene and its derivatives forming exciplex peaks with different compounds have been reported in the literature.67-70 The peak at 470 nm is absent in pure HPMC films when pyrene was added at the same concentration as in the ASD films, thus eliminating the possibility of concentration related appearance of the exciplex peak. These observations indicate that the exciplex peak in the ASD films is indeed due to the presence of probe molecules in the drug-rich domains of the films. If the films were comprised of a single miscible phase, the pyrene molecules would be uniformly dispersed in the ASD matrix and the distance between two pyrene molecules or between ITZ and pyrene would most likely be greater than the required distance of ~10Å (this supposition is based on the absence of excimer peak in HPMC films when pyrene was used in same concentration as in the ITZ-HPMC solutions) thus giving a spectrum lacking an exciplex peak. Immediately after preparation, the films were translucent to opaque. Typically, molecularly miscible systems give rise to transparent films upon solvent evaporation and drying,71 so this observation supports the postulated phase separation. When the films were heated at 165°C, they became transparent. The change in the optical properties of the films from translucent to transparent may indicate heat induced mixing at all the ratios studied. To confirm this hypothesis, fluorescence spectra of the films containing pyrene were acquired after heating at 165°C and keeping the temperature constant for 2 min (prodan is not stable at high

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Molecular Pharmaceutics

temperatures). As evident form the results (Table 1) following heating, the I/III ratio increases, suggesting that the probe molecules are registering a more hydrophilic environment than prior to heating, suggesting mixing of HPMC and ITZ. The pyrene I/III ratio is known to increase with an increase in polarity of the pyrene environment.51, 52 The increase in the transparency of the films and the increase in the I/III ratio of pyrene suggest that heat-induced mixing is occurring in the ASD films. As noted earlier, the films showed a pyrene exciplex peak suggesting that the pyrene molecules are in close proximity to ITZ. When heated at 165°C, the exciplex peak disappeared (Figure 2b), indicating that the increased interaction between ITZ and HPMC (heat induced miscibility) leads to the disruption of excited state interactions between pyrene and ITZ. The higher I/III ratios, together with the disappearance of the exciplex peak and increased transparency of the films indicate that the extent of miscibility increases upon heating. When MeOH:THF was used as the solvent system for preparing 50:50 ITZ:HPMC ASD films, the I/III pyrene peak ratio was 1.39 which is similar to the ratio observed for 50:50 films prepared using MeOH:DCM, suggesting that phase separation also occurred when the solvent system was altered. Fluorescence Imaging ITZ-HPMC films containing the fluorescent probe, pyrene, were imaged under a high-resolution fluorescence microscope. If the system shows phase separation, and the fluorescent probe has a higher affinity for the drug-rich phase (which is expected based on the hydrophobicity of the probes employed), then the drug-rich domains will be highly fluorescent when exposed to radiation of an appropriate excitation wavelength. Figure 3a shows a representative fluorescence image of 10:90 ITZ:HPMC film. The non-homogenous nature of the films, even at very low drug loading, is clearly evident with the discreet drug-rich domains showing a higher fluorescence

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intensity than the continuous phase. Similar phase separated images were observed for all the drug-polymer ratios studied. After heating at 165°C, the fluorescence intensity is more uniform suggesting some level of mixing between the drug and the polymer at high temperature (Figure 3b). The results are in accordance with those obtained using fluorescence spectroscopy. Differential Scanning Calorimetry A single calorimetric glass transition event (Tg) for an ASD is generally considered as an indicator of drug-polymer miscibility. Figure 4 shows the reversing heat flow signal for the ITZHPMC ASDs prepared by rotary evaporation. Table 2 lists the onset, midpoint and offset Tg values of all the ASDs studied. The midpoint Tg values for ITZ alone and HPMC alone were 60 °C and 140 °C respectively (data not shown). It can be seen that the dispersions show a single midpoint Tg value for all the ratios studied along with the expected increase in the overall Tg values with increasing polymer amounts in the ASD. The DSC results indicate that a miscible system is formed between ITZ and HPMC at all the ratios under investigation. From Table 2 and Figure 4, it becomes obvious that the glass transition is a broad event for all the drug-polymer ratios studied. After reheating the dispersions, the results indicate that the difference between the onset and offset Tg value decreases and the midpoint Tg decreases to a lower value i.e. the glass transition event becomes narrower. It should be noted that all the ASDs still show only one Tg in the DSC scan. The narrowing of the Tg following a heating cycle helps support our previous observations of heat-induced miscibility. When a drug and a polymer have partial miscibility, remixing of the two phases can occur during heating. The narrowing of glass transition event after heating indicates that there is less compositional variation in the system and points towards the formation of an ASD system that is relatively more miscible. AFM and nanoIR

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Figure 5 shows the bulk IR spectra of amorphous ITZ and HPMC which shows peaks that chemically discriminate the species to be identified. The peak at 1700 cm-1, arising from the carbonyl group of amorphous ITZ, is highly characteristic and can be used to distinguish between the ITZ-rich phase and the ITZ-lean phase in the films. Figure 6 shows the topographical AFM images and the corresponding IR absorption spectra from different locations in films of various ITZ-HPMC ratios before heating, whereas Figure 7 shows the same plots after heating the films at 165°C. The topographical AFM images show the existence of phase separated domains for all the drug-polymer ratios studied. However, it is not possible to obtain chemical information about the predominant component in each phase with AFM. This limitation can be overcome by using the in-built nanoscale IR methodology. The spectra from the discreet domains show the presence of a carbonyl peak indicating that these are the ITZ-rich phase whereas the continuous phase either lacks the carbonyl peak or has a very low signal indicating that it is the drug-lean phase. The results obtained from AFM-nanoIR are in excellent agreement with those obtained using fluorescence spectroscopy and fluorescence imaging and provide confirmation of our supposition that the ITZ-HPMC system is not uniformly mixed, not even at high polymer loadings. After heating, the film topography becomes more homogenous and the IR signal arising from the ITZ carbonyl peak can be found throughout the sample with almost equal intensity, supporting that heating-induced mixing occurs. AFM-nanoTA AFM together with nanoscale thermal analysis has been previously used to differentiate polymorphs72 and to study glass transition events in ASDs and polymeric layers.73, 74 It can be useful to study thermal events not easily detectable using DSC. Figure 8 shows the AFM image and the thermal traces acquired from the discrete domains and the continuous phase of the 40:60

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ITZ:HPMC films. A dip in the deflection versus temperature curve shows a thermal transition or softening point of the phase being tested. In amorphous phases, this softening point corresponds to the glass transition temperature (Tg). The ITZ rich domains show a Tg of 71 ºC which is close to the Tg of pure amorphous ITZ (Tg=60 ºC) whereas the continuous drug lean phase shows a higher Tg value of 108 ºC which indicates that this phase is polymer-rich. DISCUSSION In order to provide maximum physical stability against crystallization, it is important that the drug and polymer are miscible at the molecular level. A phase separated dispersion, where drug and polymer concentrations vary with location, will be more prone to crystallization during storage and during dissolution, which in turn will negatively impact the in vivo performance of the ASD. Consequently, it is essential to have suitable analytical methods for the assessment of miscibility in different drug-polymer dispersions. The results of this study clearly indicate that ITZ and HPMC are phase separated over the concentration range studied (i.e. 10-50% drug loading) when prepared using a solvent removal technique and evaluated using fluorescence spectroscopy or AFM-IR analysis. DSC analysis, however, was not able to show clear evidence of phase separation. The combination of IR spectroscopy with the AFM measurement further reveals that the lower viscosity component, ITZ, forms discrete drug-rich domains of 500-1000nm, dispersed in a drug-lean phase. The tendency of the lower viscosity phase to form discrete domains has been noted previously.40 The drug-rich domains are lost following heating, and ITZ becomes more uniformly dispersed within the polymer, indicating that heating promotes mixing in this system; phase separated systems have been observed to undergo mixing when heated.30, 35 The heat-induced mixing explains why the DSC results suggest that the system is miscible based on the observation of a single Tg event.

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Molecular Pharmaceutics

The system most likely undergoes mixing during the heating encountered during the DSC scan, leading to a single, albeit broadened Tg event. There have been several instances where DSC has provided ambiguous results for miscibility analysis, including for the HPMC-ITZ system studied herein.36, 59 Flory Huggins (FH) theory has been widely used to evaluate the thermodynamics of drugpolymer mixing and to understand the miscibility behavior for ASDs.75, 76 Equation 1 represents the FH equation for the free energy of mixing for a binary system containing a drug and a polymer. The equation can be deconvoluted into entropic and enthalpic effects due to mixing of the two components. In this equation, ∆Gmix is the free energy of mixing, R is the universal gas constant, T is the temperature in K, the subscripts D and P stand for drug and polymer respectively, n is the number of moles, ϕ is the volume fraction and χ is the interaction parameter or the FH parameter.

∆ 

= ∅ +  ∅ + ∅ 

Equation 1

The first two terms on the right hand side of the equation represent the entropic contributions towards the free energy of mixing and are always favorable towards reducing the free energy as mixing leads to increased randomness in the system. The third term represents the non-ideality of mixing including the enthalpic contribution, which in turn depends on the type and extent of the interactions formed between the drug and the polymer. The χ parameter thus provides a measure of how favorable the adhesive interactions are between the two components undergoing mixing relative to the cohesive interactions in the pure components. Thus, a negative value of the χ parameter implies a favorable free energy of mixing as a result of stronger adhesive interactions between the drug and the polymer molecules, than the cohesive interactions of the pure components.77 A positive value of interaction parameter χ denotes stronger cohesive interactions

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and poor drug-polymer interactions, indicating that the change in the entropy of mixing should be large enough to overcome the unfavorable enthalpy changes in order to result in a single phase miscible system. Evaluating the molecular structure of the ASD components, it is apparent that HPMC has substituted functional groups that decrease the total number of H-bond donating hydroxyl groups available to H-bond with the H-bond acceptor carbonyl group of ITZ while ITZ has no H-bond donor groups to interact with the acceptor groups on HPMC. Hence, based on the chemistry of ITZ and HPMC (figure 1), any miscibility between the two components is likely to be entropically driven, with little contribution from favorable intermolecular interactions. 20 The drug-polymer system under investigation has been studied previously in terms of miscibility by Six et al and it was found to be phase separated at higher drug loadings (40%) using thermal analysis.59 The findings reported in this study are in agreement with this report and also provide a detailed analysis of the microstructure of the obtained ASDs along with the impact of different drug loadings on the miscibility. The phase separation phenomenon observed in the ITZ-HPMC ASDs could be the result of a kinetic process, namely solvent-induced immiscibility or it could have a thermodynamic origin whereby the free energy of mixing is unfavorable at room temperature. Several cases of phase separation due to solvent effects have been previously documented for both polymeric78,

79

and pharmaceutical systems.20,

21, 30

Solvent-induced

immiscibility could be even more pronounced when a mixed solvent system is used, whereby the solvents have different boiling points and the solubility of the ASD components in each solvent varies. In the present case, ITZ is freely soluble in DCM and poorly soluble in MeOH whereas HPMC is soluble in a mixture of MeOH and DCM. Thus as the solvent amount decreases in the evaporation step with DCM being preferentially lost due to its higher volatility, ITZ might reasonably be expected to phase separate as it exceeds its miscibility limit with MeOH, leading

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to formation of drug-rich and polymer-rich domains throughout the ASD matrix, as observed experimentally. The midpoint Tg values of amorphous ITZ and HPMC are 60 ˚C and 140 ˚C respectively. Thus at room temperature, after complete removal of solvents, the phase separated domains will be “glassy” in nature leading to a kinetically trapped system. Hence, even if the ITZ-HPMC system has favorable mixing thermodynamics in the absence of solvent, the mixing kinetics at room temperature will be very slow, and hence a phase separated system would be expected to persist. However, heating above the Tg would reduce the mobility constraints, enabling the system to progress towards the thermodynamically favored miscible state. An alternative explanation for the observed phase separation and heat-induced remixing is that ITZ and HPMC have limited thermodynamic miscibility at room temperature, but that the extent of mixing increases with temperature. Therefore, when prepared at room temperature by solvent evaporation, phase separation is observed. However, at high temperatures close to the melting point of the drug, mixing is thermodynamically favorable due to the increased entropy contribution, and the drug and the polymer have sufficiently high mobility so that mixing can occur. Hence mixing occurs on heating and, upon cooling, the system is kinetically trapped in a miscible state due to the reduced molecular mobility. Heat-induced miscibility can be understood by examining the phase diagram of a drug-polymer blend with an upper critical solution temperature (UCST) as shown in figure 9. Above a certain critical temperature, Tc, a molecularly miscible dispersion of the drug in the polymer will be formed at all compositions.77 Thus, even if mixing is not favored at room temperature, a miscible system can still be obtained at elevated temperatures because of the altered thermodynamics. In the case of ITZ-HPMC films, cooling of the films to room temperature will very likely trap the glassy system in a miscible state where the kinetics of de-mixing are prohibitively slow. Figure 9 shows a schematic of the mechanisms that

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can take place when an ITZ-HPMC film is formed and heated. Unfortunately, it is very hard to determine if the origin of the phase separation observed in the ITZ-HPMC blends has thermodynamic or kinetic origins. Implications of Observed Behavior on Product Processing and Stability The two most commonly employed methods for the preparation of ASDs are solvent evaporation i.e. spray drying/spray coating and melt mixing i.e. hot melt extrusion. Processing conditions or manufacturing methods can have a significant impact on the final products obtained which in turn may lead to very different performance and/or stability profile, as noted for ASDs prepared via. spray drying and hot melt extrusion.80-82 Based on the observations made in this study, spray drying with a similar solvent system might lead to some degree of phase separation in an ITZHPMC dispersion, while HME of the same system would be more likely to yield a dispersion where the drug is homogenously dispersed in the polymer, provided mixing efficiency in the extruder is not the limiting factor.59, 83 Interestingly, commercial formulations of itraconazole solid dispersions, formulated with HPMC, are available as both spray dried (Sporanox®) and hot melt extruded (OnmelTM) products. The microstructure of these products is not currently known. In terms of product performance, a partially miscible ASD with drug- rich domains would be expected to have an increased tendency towards crystallization as the crystallization inhibitory effect of the polymer is reduced due to the phase separation and hence decreased concentration in the vicinity of the drug molecules. Also, the dissolution properties may also be altered since the polymer-rich phase will dissolve quickly leaving behind the drug-rich phase which will have poorer wettability and slower dissolution characteristics. Clearly, in order to elucidate the impact of processing on the resultant ASD microstructure, appropriate analytical methodologies are essential that enable characterization of compositional and spatial variations. The AFM-nanoIR

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and fluorescence techniques described herein are therefore useful additions to the toolbox of techniques used for ASD characterization. CONCLUSIONS The microstructure and miscibility of itraconazole-HPMC amorphous solid dispersions was assessed using various orthogonal techniques that provided information about the size and composition of heterogeneous domains. When prepared using solvent evaporation, phase separation was observed with the formation of discrete itraconazole-rich domains dispersed in an HPMC continuous phase. Both AFM-nanoIR spectroscopy and fluorescence imaging were able to provide information about the microstructure of the ASD prepared by solvent evaporation. The use of environment sensitive fluorescence probes provided further information about the extent of miscibility in the blends, while DSC analysis provided ambiguous results. Heating was found to promote mixing between ITZ and HPMC, with a concurrent loss of structural and chemical heterogeneity. These new analytical approaches offer promise for investigating process-induced variations in ASD structure.

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FIGURES

Figure 1. Structures of the compounds used. Prodan (a), Pyrene (b), HPMC (c) and ITZ (d).

Figure 2. Emission spectra of pyrene in an ITZ-HPMC film with 10% drug loading. Figure 2a corresponds to the spectrum before heating whereas figure 2b corresponds to the emission spectrum after heating at 165˚C. The ratio of peak I to peak III increases following heating. Also note the disappearance of the exciplex peak at 475 nm in the spectrum after heating.

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Figure 3. Images of 10:90 ITZ-HPMC ASD films containing pyrene before (a) and after (b) heating acquired using a fluorescent microscope. Scale bar corresponds to 20µm.

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Figure 4. DSC thermograms of powdered ASDs of various ITZ-HPMC ratios during first (a) and (b) second heating cycles.

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Amorphous ITZ HPMC

Absorbance

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2000

1800

1600

1400

1200

1000

800

600

Wavenumber (cm-1) Figure 5. Bulk IR spectra of amorphous ITZ and HPMC. The amorphous ITZ peak at 1700 cm-1 (indicated by arrow) was used to differentiate drug-rich and drug-lean phases in ASD films.

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Figure 6. AFM topographic images (left) and nanoscale spectra (right) obtained from the ITZHPMC ASD films before heating. The drug-polymer ratios were 10:90 (a), 20:80 (b), 30:70 (c), 40:60 (d) and 50:50 (e). The markers on the topography images show the point from which the IR spectrum was collected whereby the blue markers correspond to the blue spectra, and the red markers to the red spectra. The color scale on the topography image shows the Z-height variation of the sample.

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Figure 7. AFM topographic images (left) and nanoscale spectra (right) obtained from the ITZHPMC ASD films after heating at 165˚C. The drug-polymer ratios were 10:90 (a), 20:80 (b), 30:70 (c), 40:60 (d) and 50:50 (e). The markers on the topography images show the locations from which the IR spectrum was collected. The color scale on the topography image shows the Z-height of the sample.

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Figure 8. AFM topographical (left) and deflection (right) images obtained from a 40:60 ITZ:HPMC film. The deflection versus temperature plot represents the Tg of the different phases (dip in the curve). Green curve is from the drug rich domain and the purple curves are from the drug lean continuous phase.

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Figure 9. Typical phase diagram of a drug-polymer blend showing upper critical solution temperature (UCST) behavior.

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Figure 10. Schematic showing the possible mechanisms contributing to the (im)miscibility of ITZ-HPMC ASDs.

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TABLES Table 1. Wavelength maximum of prodan and I/III ratio of pyrene in ITZ-HPMC films before and after heating at 165 ˚C. Prodan was not stable at high temperatures. The standard deviation in I/III ratio for pyrene containing films was less than 0.02 and for prodan containing samples, it was less than 2 nm. Before heating Drug-polymer HPMC ITZ 50:50 40:60 30:70 20:80 10:90

Prodan (nm) 452.9 439.0 439.3 438.7 439.3 440.7 443.7

Pyrene (I/III) 1.54 1.39 1.39 1.40 1.41 1.42 1.42

After heating Pyrene (I/III) --1.50 1.50 1.53 1.50 1.52

Table 2. Onset, midpoint and offset values of the glass transition event in various ITZ-HPMC ASDs in first and second heating scans. Drugpolymer ratio

Onset (˚C) 1st run

Midpoint (˚C)

2nd run 1st run 2nd run

Offset (˚C) 1st run

2nd run

50:50 40:60 30:70

70 78 84

62 66 73

77 87 100

64 70 80

81 95 108

71 76 86

20:80

94

84

116

90

119

100

10:90

112

110

131

115

135

116

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AUTHOR INFORMATION Corresponding author *Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, Unites States of America. Tel: (765) 4966614. Fax: (765) 494-6545. E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS The authors would like to thank the National Science Foundation through grant numbers EEC0540855 and IIP- 1152308 and the National Institutes of Health through grant numbers R41 GM100657-01A1 and R42 GM100657-03, for financial support. Aaron J. Harrison is thanked for providing training on the AFM instrument.

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REFERENCES 1. Hancock, B. C.; Parks, M. What is the true solubility advantage for amorphous pharmaceuticals? Pharm. Res. 2000, 17, (4), 397-404. 2. Hancock, B. C.; Zografi, G. Characteristics and significance of the amorphous state in pharmaceutical systems. J. Pharm. Sci. 1997, 86, (1), 1-12. 3. Newman, A.; Knipp, G.; Zografi, G. Assessing the performance of amorphous solid dispersions. J. Pharm. Sci. 2012, 101, (4), 1355-1377. 4. Di, L.; Fish, P. V.; Mano, T. Bridging solubility between drug discovery and development. Drug Discovery Today 2012, 17, (9), 486-495. 5. Lipinski, C. A. Poor aqueous solubility-an industry wide problem in ADME screening. Am. Pharm. Rev. 2002, 5, 82-85. 6. Janssens, S.; Van den Mooter, G. Review: physical chemistry of solid dispersions. J. Pharm. Pharmacol. 2009, 61, (12), 1571-1586. 7. Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 2013, 65, (1), 315-499. 8. Rumondor, A. C.; Stanford, L. A.; Taylor, L. S. Effects of polymer type and storage relative humidity on the kinetics of felodipine crystallization from amorphous solid dispersions. Pharm. Res. 2009, 26, (12), 2599-2606. 9. Janssens, S.; De Zeure, A.; Paudel, A.; Van Humbeeck, J.; Rombaut, P.; Van den Mooter, G. Influence of preparation methods on solid state supersaturation of amorphous solid dispersions: a case study with itraconazole and eudragit e100. Pharm. Res. 2010, 27, (5), 775785. 10. Paudel, A.; Geppi, M.; Van den Mooter, G. Structural and Dynamic Properties of Amorphous Solid Dispersions: The Role of Solid ‐ State Nuclear Magnetic Resonance Spectroscopy and Relaxometry. J. Pharm. Sci. 2014, 103, (9), 2635-2662. 11. Vasanthavada, M.; Tong, W.-Q. T.; Joshi, Y.; Kislalioglu, M. S. Phase behavior of amorphous molecular dispersions II: Role of hydrogen bonding in solid solubility and phase separation kinetics. Pharm. Res. 2005, 22, (3), 440-448. 12. Brettmann, B. K.; Myerson, A. S.; Trout, B. L. Solid‐state nuclear magnetic resonance study of the physical stability of electrospun drug and polymer solid solutions. J. Pharm. Sci. 2012, 101, (6), 2185-2193. 13. Qi, S.; Moffat, J. G.; Yang, Z. Early stage phase separation in pharmaceutical solid dispersion thin films under high humidity: improved spatial understanding using probe-based thermal and spectroscopic nanocharacterization methods. Mol. Pharmaceutics 2013, 10, (3), 918-930. 14. Galop, M. Study of pharmaceutical solid dispersions by microthermal analysis. Pharm. Res. 2005, 22, (2), 293-302. 15. Qi, S.; Belton, P.; Nollenberger, K.; Gryczke, A.; Craig, D. Q. Compositional analysis of low quantities of phase separation in hot-melt-extruded solid dispersions: a combined atomic force microscopy, photothermal fourier-transform infrared microspectroscopy, and localised thermal analysis approach. Pharm. Res. 2011, 28, (9), 2311-2326. 16. Six, K.; Murphy, J.; Weuts, I.; Craig, D. Q.; Verreck, G.; Peeters, J.; Brewster, M.; Van den Mooter, G. Identification of phase separation in solid dispersions of itraconazole and Eudragit® E100 using microthermal analysis. Pharm. Res. 2003, 20, (1), 135-138.

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Page 34 of 39

17. Ivanisevic, I. Physical stability studies of miscible amorphous solid dispersions. J. Pharm. Sci. 2010, 99, (9), 4005-4012. 18. Ivanisevic, I.; Bates, S.; Chen, P. Novel methods for the assessment of miscibility of amorphous drug‐polymer dispersions. J. Pharm. Sci. 2009, 98, (9), 3373-3386. 19. Newman, A.; Engers, D.; Bates, S.; Ivanisevic, I.; Kelly, R. C.; Zografi, G. Characterization of amorphous API: polymer mixtures using X‐ray powder diffraction. J. Pharm. Sci. 2008, 97, (11), 4840-4856. 20. Rumondor, A. C.; Wikström, H.; Van Eerdenbrugh, B.; Taylor, L. S. Understanding the tendency of amorphous solid dispersions to undergo amorphous–amorphous phase separation in the presence of absorbed moisture. AAPS PharmSciTech 2011, 12, (4), 1209-1219. 21. Rumondor, A. C.; Taylor, L. S. Effect of polymer hygroscopicity on the phase behavior of amorphous solid dispersions in the presence of moisture. Mol. Pharmaceutics 2010, 7, (2), 477-490. 22. Marsac, P. J.; Konno, H.; Rumondor, A. C.; Taylor, L. S. Recrystallization of nifedipine and felodipine from amorphous molecular level solid dispersions containing poly (vinylpyrrolidone) and sorbed water. Pharm. Res. 2008, 25, (3), 647-656. 23. Kim, K. H.; Frank, M. J.; Henderson, N. L. Application of differential scanning calorimetry to the study of solid drug dispersions. J. Pharm. Sci. 1985, 74, (3), 283-289. 24. Van den Mooter, G.; Wuyts, M.; Blaton, N.; Busson, R.; Grobet, P.; Augustijns, P.; Kinget, R. Physical stabilisation of amorphous ketoconazole in solid dispersions with polyvinylpyrrolidone K25. Eur. J. Pharm. Sci. 2001, 12, (3), 261-269. 25. Van Drooge, D.; Hinrichs, W.; Visser, M.; Frijlink, H. Characterization of the molecular distribution of drugs in glassy solid dispersions at the nano-meter scale, using differential scanning calorimetry and gravimetric water vapour sorption techniques. Int. J. Pharm. 2006, 310, (1), 220-229. 26. Yoshihashi, Y.; Iijima, H.; Yonemochi, E.; Terada, K. Estimation of physical stability of amorphous solid dispersion using differential scanning calorimetry. J. Therm. Anal. Calorim. 2006, 85, (3), 689-692. 27. Chappell, J.; Lidzey, D. G.; Jukes, P. C.; Higgins, A. M.; Thompson, R. L.; O'Connor, S.; Grizzi, I.; Fletcher, R.; O'Brien, J.; Geoghegan, M. Correlating structure with fluorescence emission in phase-separated conjugated-polymer blends. Nat. Mater. 2003, 2, (9), 616-621. 28. Lauer, M. E.; Siam, M.; Tardio, J.; Page, S.; Kindt, J. H.; Grassmann, O. Rapid assessment of homogeneity and stability of amorphous solid dispersions by atomic force microscopy—from bench to batch. Pharm. Res. 2013, 30, (8), 2010-2022. 29. Qi, S.; Belton, P.; Nollenberger, K.; Clayden, N.; Reading, M.; Craig, D. Q. Characterisation and prediction of phase separation in hot-melt extruded solid dispersions: a thermal, microscopic and NMR relaxometry study. Pharm. Res. 2010, 27, (9), 1869-1883. 30. Marsac, P. J.; Rumondor, A. C.; Nivens, D. E.; Kestur, U. S.; Stanciu, L.; Taylor, L. S. Effect of temperature and moisture on the miscibility of amorphous dispersions of felodipine and poly (vinyl pyrrolidone). J. Pharm. Sci. 2010, 99, (1), 169-185. 31. Padilla, A. M.; Chou, S. G.; Luthra, S.; Pikal, M. J. The study of amorphous phase separation in a model polymer phase‐separating system using raman microscopy and a low‐ temperature stage: Effect of cooling rate and nucleation temperature. J. Pharm. Sci. 2011, 100, (4), 1362-1376. 32. Padilla, A. M.; Ivanisevic, I.; Yang, Y.; Engers, D.; Bogner, R. H.; Pikal, M. J. The study of phase separation in amorphous freeze ‐ dried systems. part I: Raman mapping and

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computational analysis of XRPD data in model polymer systems. J. Pharm. Sci. 2011, 100, (1), 206-222. 33. Padilla, A. M.; Pikal, M. J. The study of phase separation in amorphous freeze‐dried systems, part 2: Investigation of raman mapping as a tool for studying amorphous phase separation in freeze‐dried protein formulations. J. Pharm. Sci. 2011, 100, (4), 1467-1474. 34. Van Eerdenbrugh, B.; Taylor, L. S. Application of mid-IR spectroscopy for the characterization of pharmaceutical systems. Int. J. Pharm. 2011, 417, (1), 3-16. 35. Baird, J. A.; Taylor, L. S. Evaluation of amorphous solid dispersion properties using thermal analysis techniques. Adv. Drug Delivery Rev. 2012, 64, (5), 396-421. 36. Qian, F.; Huang, J.; Zhu, Q.; Haddadin, R.; Gawel, J.; Garmise, R.; Hussain, M. Is a distinctive single T g a reliable indicator for the homogeneity of amorphous solid dispersion? Int. J. Pharm. 2010, 395, (1), 232-235. 37. Kjoller, K.; Felts, J.; Cook, D.; Prater, C.; King, W. High-sensitivity nanometer-scale infrared spectroscopy using a contact mode microcantilever with an internal resonator paddle. Nanotechnology 2010, 21, (18), 185705. 38. Dazzi, A.; Prazeres, R.; Glotin, F.; Ortega, J.; Al-Sawaftah, M.; De Frutos, M. Chemical mapping of the distribution of viruses into infected bacteria with a photothermal method. Ultramicroscopy 2008, 108, (7), 635-641. 39. Mayet, C.; Dazzi, A.; Prazeres, R.; Allot, F.; Glotin, F.; Ortega, J. Sub-100 nm IR spectromicroscopy of living cells. Opt. Lett. 2008, 33, (14), 1611-1613. 40. Van Eerdenbrugh, B.; Lo, M.; Kjoller, K.; Marcott, C.; Taylor, L. S. Nanoscale midinfrared evaluation of the miscibility behavior of blends of dextran or maltodextrin with poly (vinylpyrrolidone). Mol. Pharmaceutics 2012, 9, (5), 1459-1469. 41. Van Eerdenbrugh, B.; Lo, M.; Kjoller, K.; Marcott, C.; Taylor, L. S. Nanoscale mid‐ infrared imaging of phase separation in a drug–polymer blend. J. Pharm. Sci. 2012, 101, (6), 2066-2073. 42. Bains, G.; Patel, A. B.; Narayanaswami, V. Pyrene: a probe to study protein conformation and conformational changes. Molecules 2011, 16, (9), 7909-7935. 43. Cabanelas, J.; Serrano, B.; González‐Benito, J.; Bravo, J.; Baselga, J. Morphology of epoxy/polyorganosiloxane reactive blends. Macromol. Rapid Commun. 2001, 22, (9), 694-699. 44. Miller, K. E.; Krueger, R. H.; Torkelson, J. M. Mobility‐sensitive fluorescence probes for quantitative monitoring of water sorption and diffusion in polymer coatings. J. Polym. Sci., Part B: Polym. Phys. 1995, 33, (17), 2343-2349. 45. Okay, O.; Kaya, D.; Pekcan, O. Free-radical crosslinking copolymerization of styrene and divinylbenzene: real time monitoring of the gel effect using fluorescence probe. Polymer 1999, 40, (22), 6179-6187. 46. Serrano, B.; Baselga, J.; Bravo, J.; Mikes, F.; Sese, L.; Esteban, I.; Pierola, I. Chemical imaging of phase-separated polymer blends by fluorescence microscopy. J. Fluoresc. 2000, 10, (2), 135-135. 47. Strehmel, B.; Strehmel, V.; Younes, M. Fluorescence probes for investigation of epoxy systems and monitoring of crosslinking processes. J. Polym. Sci., Part B: Polym. Phys. 1999, 37, (13), 1367-1386. 48. Ilevbare, G. A.; Taylor, L. S. Liquid–liquid phase separation in highly supersaturated aqueous solutions of poorly water-soluble drugs: Implications for solubility enhancing formulations. Cryst. Growth Des. 2013, 13, (4), 1497-1509.

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49. Jackson, M. J.; Toth, S. J.; Kestur, U. S.; Huang, J.; Qian, F.; Hussain, M. A.; Simpson, G. J.; Taylor, L. S. Impact of polymers on the precipitation behavior of highly supersaturated aqueous danazol solutions. Mol. Pharmaceutics 2014, 11, (9), 3027-3038. 50. Purohit, H. S.; Taylor, L. S. Phase Separation Kinetics in Amorphous Solid Dispersions Upon Exposure to Water. Mol. Pharmaceutics 2015, 12, (5), 1623-1635. 51. Ananthapadmanabhan, K.; Goddard, E.; Turro, N.; Kuo, P. Fluorescence probes for critical micelle concentration. Langmuir 1985, 1, (3), 352-355. 52. Kalyanasundaram, K.; Thomas, J. Environmental effects on vibronic band intensities in pyrene monomer fluorescence and their application in studies of micellar systems. J. Am. Chem. Soc. 1977, 99, (7), 2039-2044. 53. Kalyanasundaram, K.; Thomas, J. K. Solvent-dependent fluorescence of pyrene-3carboxaldehyde and its applications in the estimation of polarity at micelle-water interfaces. J. Phys. Chem. 1977, 81, (23), 2176-2180. 54. Fukuda, R.; Chidthong, R.; Cammi, R.; Ehara, M. Optical absorption and fluorescence of PRODAN in solution: Quantum chemical study based on the symmetry-adapted clusterconfiguration interaction method. Chem. Phys. Lett. 2012, 552, 53-57. 55. Parusel, A. B.; Schneider, F. W.; Köhler, G. An ab initio study on excited and ground state properties of the organic fluorescence probe PRODAN. J. Mol. Struct.: THEOCHEM 1997, 398, 341-346. 56. Catalan, J.; Perez, P.; Laynez, J.; Blanco, F. G. Analysis of the solvent effect on the photophysics properties of 6-propionyl-2-(dimethylamino) naphthalene (PRODAN). J. Fluoresc. 1991, 1, (4), 215-223. 57. Krasnowska, E. K.; Gratton, E.; Parasassi, T. Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases. Biophys. J. 1998, 74, (4), 1984-1993. 58. Janssens, S.; Roberts, C.; Smith, E. F.; Van den Mooter, G. Physical stability of ternary solid dispersions of itraconazole in polyethyleneglycol 6000/hydroxypropylmethylcellulose 2910 E5 blends. Int. J. Pharm. 2008, 355, (1), 100-107. 59. Six, K.; Berghmans, H.; Leuner, C.; Dressman, J.; Van Werde, K.; Mullens, J.; Benoist, L.; Thimon, M.; Meublat, L.; Verreck, G. Characterization of solid dispersions of itraconazole and hydroxypropylmethylcellulose prepared by melt extrusion, part II. Pharm. Res. 2003, 20, (7), 1047-1054. 60. Six, K.; Daems, T.; de Hoon, J.; Van Hecken, A.; Depre, M.; Bouche, M.-P.; Prinsen, P.; Verreck, G.; Peeters, J.; Brewster, M. E. Clinical study of solid dispersions of itraconazole prepared by hot-stage extrusion. Eur. J. Pharm. Sci. 2005, 24, (2), 179-186. 61. Marcott, C.; Lo, M.; Kjoller, K.; Prater, C.; Noda, I. Spatial differentiation of submicrometer domains in a poly (hydroxyalkanoate) copolymer using instrumentation that combines atomic force microscopy (AFM) and infrared (IR) spectroscopy. Appl. Spectrosc. 2011, 65, (10), 1145-1150. 62. Dong, D. C.; Winnik, M. A. The Py scale of solvent polarities. Can. J. Chem. 1984, 62, (11), 2560-2565. 63. Lissi, E.; Abuin, E.; Rubio, M.; Ceron, A. Fluorescence of Prodan and Laurdan in AOT/heptane/water microemulsions: partitioning of the probes and characterization of microenvironments. Langmuir 2000, 16, (1), 178-181. 64. Dunn, B.; Zink, J. I. Probes of pore environment and molecule-matrix interactions in solgel materials. Chem. Mater. 1997, 9, (11), 2280-2291.

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65. Verin, A. D.; Gusev, N. B. Ca 2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene) maleimide fluorescence. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1988, 956, (2), 197-208. 66. Liu, G.; Guillet, J. A kinetic description of diffusion-controlled intramolecular excimer and exciplex formation. Macromolecules 1990, 23, (19), 4292-4298. 67. Cao, T.; Munk, P.; Ramireddy, C.; Tuzar, Z.; Webber, S. Fluorescence studies of amphiphilic poly (methacrylic acid)-block-polystyrene-block-poly (methacrylic acid) micelles. Macromolecules 1991, 24, (23), 6300-6305. 68. Hinatu, J.; Masuhara, H.; Mataga, N.; Sakata, Y.; Misumi, S. Absorption spectra of interand intramolecular exciplex systems of pyrene and N, N-dimethylaniline in alcoholic solutions. Bull. Chem. Soc. Jpn. 1978, 51, (4), 1032-1036. 69. Katusin-Razem, B.; Wong, M.; Thomas, J. K. The effect of micellar phase on the state and dynamics of some excited state charge transfer complexes. J. Am. Chem. Soc. 1978, 100, (6), 1679-1686. 70. Palmans, J.; Van der Auweraer, M.; Swinnen, A.; De Schryver, F. Intermolecular exciplex formation between pyrene derivatives and 1, 2-dimethylindole. J. Am. Chem. Soc. 1984, 106, (25), 7721-7728. 71. Feng, J.; Winnik, M. A.; Shivers, R. R.; Clubb, B. Polymer blend latex films: morphology and transparency. Macromolecules 1995, 28, (23), 7671-7682. 72. Sanders, G.; Roberts, C.; Danesh, A.; Murray, A.; Price, D.; Davies, M.; Tendler, S.; Wilkins, M. Discrimination of polymorphic forms of a drug product by localized thermal analysis. J. Microsc. 2000, 198, (2), 77-81. 73. Maruf, S. H.; Ahn, D. U.; Greenberg, A. R.; Ding, Y. Glass transition behaviors of interfacially polymerized polyamide barrier layers on thin film composite membranes via nanothermal analysis. Polymer 2011, 52, (12), 2643-2649. 74. Zhang, J.; Bunker, M.; Chen, X.; Parker, A. P.; Patel, N.; Roberts, C. J. Nanoscale thermal analysis of pharmaceutical solid dispersions. Int. J. Pharm. 2009, 380, (1), 170-173. 75. Tian, Y.; Booth, J.; Meehan, E.; Jones, D. S.; Li, S.; Andrews, G. P. Construction of drug – polymer thermodynamic phase diagrams using Flory – Huggins interaction theory: identifying the relevance of temperature and drug weight fraction to phase separation within solid dispersions. Mol. Pharmaceutics 2012, 10, (1), 236-248. 76. Tian, Y.; Caron, V.; Jones, D. S.; Healy, A. M.; Andrews, G. P. Using Flory–Huggins phase diagrams as a pre‐formulation tool for the production of amorphous solid dispersions: a comparison between hot‐melt extrusion and spray drying. J. Pharm. Pharmacol. 2014, 66, (2), 256-274. 77. Bates, F. S. Polymer-polymer phase behavior. Science 1991, 251, (4996), 898-905. 78. Pouchlý, J.; Patterson, D. Polymers in mixed solvents. Macromolecules 1976, 9, (4), 574-579. 79. Robard, A.; Patterson, D.; Delmas, G. The" Δχ Effect" and Polystyrene-Poly (vinyl methyl ether) Compatibility in Solution. Macromolecules 1977, 10, (3), 706-708. 80. Agrawal, A. M.; Dudhedia, M. S.; Patel, A. D.; Raikes, M. S. Characterization and performance assessment of solid dispersions prepared by hot melt extrusion and spray drying process. Int. J. Pharm. 2013, 457, (1), 71-81. 81. Guns, S.; Dereymaker, A.; Kayaert, P.; Mathot, V.; Martens, J. A.; Van den Mooter, G. Comparison between hot-melt extrusion and spray-drying for manufacturing solid dispersions of the graft copolymer of ethylene glycol and vinylalcohol. Pharm. Res. 2011, 28, (3), 673-682.

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82. Mahmah, O.; Tabbakh, R.; Kelly, A.; Paradkar, A. A comparative study of the effect of spray drying and hot ‐ melt extrusion on the properties of amorphous solid dispersions containing felodipine. J. Pharm. Pharmacol. 2014, 66, (2), 275-284. 83. DiNunzio, J. C.; Brough, C.; Miller, D. A.; Williams, R. O.; McGinity, J. W. Fusion processing of itraconazole solid dispersions by KinetiSol® dispersing: a comparative study to hot melt extrusion. J. Pharm. Sci. 2010, 99, (3), 1239-1253.

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For Table of Contents Only Miscibility of Itraconazole-Hydroxypropyl Methylcellulose Blends- Insights with High Resolution Analytical Methodologies. Authors: Hitesh S. Purohit and Lynne S. Taylor

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