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Nanoscale Infrared, Thermal, and Mechanical Characterization of Telaprevir-Polymer Miscibility in Amorphous Solid Dispersions Prepared by Solvent Evaporation Na Li, and Lynne S. Taylor Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00925 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 15, 2016

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Molecular Pharmaceutics

1 2 3

Nanoscale Infrared, Thermal, and Mechanical Characterization of Telaprevir-Polymer Miscibility

4

in Amorphous Solid Dispersions Prepared by Solvent Evaporation

5 6 Na Li† and Lynne S. Taylor†*

7 8 9 10

†

Department of Industrial and Physical Pharmacy, Purdue University, 575 Stadium Mall Drive, West Lafayette, Indiana 47907, United States

11 12

ACS Paragon Plus Environment


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TOC graphic

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nano IR spectra

nano thermal analysis

nano mechanical spectra

-2.6

Deflection (V)

-2.8

Absorbance

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TPV HPMC

-3.0 -3.2 -3.4 -3.6 -3.8

1800

15 16

1600

1400

Wavenumber (cm-1)

1200

40

60

80

100 120 140 160 180

Temperature (oC)


120

125

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Frequency (kHz)

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Abstract

18

Miscibility is of great interest for pharmaceutical systems, in particular for amorphous

19

solid dispersions, as phase separation can lead to a higher tendency to crystallize, resulting in a

20

loss in solubility, decreased dissolution rate, and compromised bioavailability. The purpose of this

21

study was to investigate the miscibility behavior of a model poorly water-soluble drug, telaprevir

22

(TPV), with three different polymers using atomic force microscopy-based infrared, thermal, and

23

mechanical analysis. Standard atomic force microscopy (AFM) imaging together with nanoscale

24

infrared spectroscopy (AFM-IR), nanoscale thermal analysis (nanoTA), and Lorentz contact

25

resonance (LCR) measurements were used to evaluate the miscibility behavior of TPV with three

26

polymers, hydroxypropyl methylcellulose (HPMC), HPMC acetate succinate (HPMCAS), and

27

polyvinyl pyrrolidone vinyl acetate (PVPVA), at different drug to polymer ratios. Phase separation

28

was observed with HPMC and PVPVA at drug loadings above 10%. For HPMCAS, a smaller

29

miscibility gap was observed, with phase separation being observed at drug loadings higher than

30

~30-40%. The domain size of phase separated regions varied from below 50nm to a few hundred

31

nanometers. Localized infrared spectra, nano-TA measurements, and images from AFM-based IR,

32

and LCR measurements showed clear contrast between the continuous and discrete domains for

33

these phase-separated systems whereby the discrete domains were drug-rich. Fluorescence

34

microscopy provided additional evidence for phase separation. These methods appear to be

35

promising to evaluate miscibility in drug-polymer systems with similar Tgs and sub-micron

36

domain sizes. Furthermore, such findings are of obvious importance in the context of contributing

37

to a mechanistic understanding of amorphous solid dispersion phase behavior.

38 39

Keywords: AFM, nanoIR, nanoTA, LCR, amorphous solid dispersion



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40

Introduction

41

Poor aqueous solubility is a critical issue for many of the new drugs currently being

42

developed and can limit bioavailability1. To achieve adequate delivery to the body, various

43

solubility enhancing approaches can be used2. Using an amorphous form of the drug, typically

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generated by creating a blend with a suitable polymer producing a system commonly referred to as

45

an amorphous solid dispersion (ASD), has emerged as an important solubility enhancing strategy

46

for low bioavailability compounds

47

dispersions (ASDs) 6. Formation of an amorphous molecular dispersion of the drug with a suitable

48

polymer can lead to more rapid dissolution in aqueous media and an increase in achievable

49

solution concentrations relative to formulations containing crystalline drug 7. In a miscible drug-

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polymer system, the presence of the high molecular weight polymer will also retard drug

51

crystallization and kinetically stabilize the system 8, whereas in a phase separated ASD system, the

52

amorphous drug may crystalize quickly leading to a loss in the solubility and bioavailability

53

advantage 9. Prevention of crystallization over the shelf-life of the product is essential in order to

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produce safe and efficacious formulations containing amorphous drug. Therefore, it is essential to

55

understand and characterize drug-polymer miscibility. Unfortunately, this is not a trivial endeavor.

56

Differential scanning calorimetry (DSC) is considered as the gold standard for miscibility

57

evaluation of pharmaceutical systems based on the presence of one (miscible) or two (phase

58

separation) glass transition (Tg) events

59

indicator of miscibility

60

similar Tg values, partially phase separated systems with a similar chemical composition in each

61

phases, and systems with very small domain sizes, it may be challenging to characterize

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miscibility via DSC

10, 11

12, 13

3-5

. Miscibility is of great importance for amorphous solid

10

. However, a single Tg value may not be a reliable

. In particular, for systems containing a drug and a polymer with

. Traditional high-resolution imaging techniques such as scanning


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electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force

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microscopy (AFM), are able to achieve the nanoscale resolution often necessary for miscibility

65

evaluations

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different phases. Solid state nuclear magnetic resonance (ssNMR) spectroscopy is an extremely

67

valuable tool to evaluate miscibility and provide some information on domain size 16, however, it

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is extremely time consuming to obtain the data. Consequently there is a need for additional

69

techniques that enable sample microstructure to be related to chemical composition in order to

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provide a mechanistic understanding of the miscibility behavior of ASD systems as a function of

71

factors such as polymer type, drug loading, environmental conditions, and processing variables.

14, 15

. However, these techniques are not able to identify the chemical composition of

72

The objective of this study was to develop new approaches to characterize the miscibility

73

behavior of amorphous solid dispersions using nanoscale characterization methods. Herein we

74

combined infrared spectroscopy, thermal analysis, and Lorentz contact resonance mechanical

75

measurements with standard AFM topographical imaging to identify phase separation in drug-

76

polymer blends. The molecular structures of the model drug, telaprevir, and polymers,

77

hydroxypropyl methylcellulose (HPMC), hydroxypropyl methylcellulose acetate succinate

78

(HPMCAS), and polyvinylpyrrolidone/vinyl acetate (PVPVA) are shown in Figure 1. Telaprevir is

79

a poorly water-soluble drug used to treat hepatitis C infections. Due to the low oral bioavailability

80

of the crystalline form, it was developed and commercialized as an amorphous solid dispersion

81

with HPMCAS, in order to achieve sufficient oral bioavailability

82

PVPVA are the most widely used polymers in commercial ASD formulations. In this study, the

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miscibility of thin films of TPV-polymer blends, prepared by solvent evaporation was investigated

84

as a function of polymer type and drug-polymer ratio using various nanoscale characterization

85

methods.


4, 17

. HPMC, HPMCAS, and


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Materials and methods

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Materials

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Telaprevir was supplied by Attix (Toronto, Ontario, Canada). HPMCAS (HF grade) was

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obtained from ShinEtsu (Tokyo, Japan). Methocel® E5 was obtained from the Dow Chemical

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Company (Midland, MI). Kollidon® VA 64 (PVPVA) was supplied by the BASF Corporation

92

(Ludwigshafen, Germany). Methanol, acetone, and dichloromethane were purchased from

93

Mallinckrodt Baker (Phillipsburg, NJ).

94 95

Methods

96

Preparation of TPV-polymer thin films

97

TPV-polymer mixtures at different TPV-polymer ratios were dissolved in 1:1 (v:v)

98

methanol : dichloromethane solutions unless otherwise specified. A total solids content of 5% (w/v)

99

was achieved for all systems.

100

Samples were then spin-coated onto 1cm x 1cm ZnS substrates (Anasys Instruments, Santa

101

Barbara, CA) using a spin coater (Chemat Technology Inc., Northridge, CA). 30-50uL of the

102

TPV-polymer solution was deposited, and the ZnS flat was spun for 6s at 50 rpm followed by 50s

103

at 3100 rpm. The spin-coated films were stored in a vacuum oven overnight at ambient

104

temperature to remove residual solvent.

105 106

Topographical imaging

107

A nanoIR2TM AFM-IR instrument (Anasys Instruments, Inc., Santa Barbara, CA) was used

108

to perform AFM topographical imaging. Contact mode NIR2 probes (Model: PR-EX-NIR2,


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Anasys Instruments, Inc., Santa Barbara, CA) were used to collect topographical images. For

110

tapping mode images, tapping mode AFM probes (Model: EX-T125) were used. A scan rate of 0.5

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Hz was used for contact mode, and 0.3 Hz was used for tapping mode. The image acquisition time

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was 8.6 minutes and 14.3 minutes at scan rates of 0.5 and 0.3 Hz, respectively, with an x and y

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resolution of 256 points. Topographical images were collected using the Analysis Studio software

114

(version 3.10.5539, Anasys Instruments, Inc., Santa Barbara, CA). The size of the height features

115

was measured using the ruler tool in the software with an average of 5 data points.

116 117

Bulk IR spectroscopy

118

Thin films of pure TPV, polymers, and TPV-polymer mixtures were prepared on KRS-5

119

windows (Harrick Scientific Corporation, Ossining, NY) by spin coating using the method

120

described previously. A Bruker Vertex 70 FTIR spectrometer was used (Bruker Co., Billerica,

121

MA). 128 scans were collected for both background and samples. IR spectra were collected and

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analyzed using OPUS software (version 7.2, Bruker Optik GmbH).

123 124

Nano IR spectroscopy

125

The nanoIR2TM AFM-IR instrument (Anasys Instruments, Inc., Santa Barbara, CA) was

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used to collect localized nanoscale mid IR spectra. Contact mode NIR2 probes (Model: PR-EX-

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NIR2, Anasys Instruments, Inc., Santa Barbara, CA) with a resonance frequency of 13±4 kHz and

128

a spring constant of 0.07-0.4 N/m were used for data collection. IR spectra were collected and

129

analyzed using Analysis Studio software (version 3.10.5539, Anasys Instruments, Inc., Santa

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Barbara, CA). The AFM-IR technique is accomplished by coupling a pulsed tunable IR source

131

with an AFM. The pulsed tunable IR source has a pulsed length of ~10 ns and can cover a broad



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range of the mid IR region. The light from this source is focused onto the tip-sample contact area.

133

When the pulsed light from the IR source is absorbed by the sample, a rapid heating/expansion of

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the sample occurs creating an impulse onto the AFM cantilever inducing an oscillation of the

135

cantilever, which is termed a “ringdown”.

136

frequencies due to excitation of the different modes of oscillation of the pinned AFM cantilever in

137

contact with the sample surface. Usually one of these modes is selected by a band pass filter. The

138

amplitude of this signal is proportional to the absorption of the sample integrated throughout the

139

thickness of the sample.

The “ringdown” will typically have multiple

140

Prior to IR spectra acquisition, IR background calibration was performed over the 1200-

141

1800 cm-1 range to normalize the signal intensity as a function of wavenumber. 5 background

142

spectra were collected and averaged. The second mode of cantilever oscillation was selected for

143

the cantilever “ringdown” signal by selecting a frequency center of 200 kHz with a frequency

144

window of 30kHz. The infrared light was centered at the probe-sample contact point using the

145

optimize function at 1450 cm-1, 1525 cm-1 and 1670 cm-1 for telaprevir. A co-average of 256x was

146

used for optimization. The power level was adjusted to be high enough to achieve a clean

147

optimization background with a distinct IR hotspot in the center (Figure S1, supporting

148

information). The optimization step was repeated four times from a 800µm x 800µm search area

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to a 50µm x 50µm search area. Then the IR focus was optimized by clicking the IR focus buttons

150

until the IR signal reached a maximum value. AFM-IR spectra were collected from 1200 to

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1800cm-1, with an interval of 4 cm-1. A co-average number of 256x was used for IR spectra

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collection. The spectral acquisition time was 2.5 minutes. The average spectra of 5 individual

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points from each phase, i.e. continuous and dispersed phases, were summed, normalized and

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plotted. For pure compounds, an average of 9 spectra were obtained. All AFM-IR spectra were


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smoothed using a Savitzky-Golay function with a polynomial order of 3 and a side point of 5 pt.

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All localized IR spectra were normalized prior to further analysis.

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For IR image scans, a co-average of 8x was used at a scanning rate of 0.1Hz. The x and y

158

resolution values used were both 256 pt. The image acquisition time was approximately 43

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minutes. IR ratio images were obtained by taking two IR images at different wavenumbers in the

160

same area. A sample frequency image was collected simultaneously by obtaining the contact

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resonant frequency signals from the cantilever “ringdown” as a function of position 18, 19.

162 163

Differential Scanning Calorimetry (DSC)

164

The glass transition temperatures (Tgs) of TPV, HPMC, HPMCAS, and PVPVA were

165

measured using a TA Q2000 DSC equipped with a refrigerated cooling accessory (TA Instruments,

166

New Castle, DE). Samples were weighted in TzeroTM aluminum pans and TzeroTM aluminum lids.

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Samples were heated to 260oC for TPV, 225oC for HPMC, and 150oC for HPMCAS and PVPVA,

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using a heating rate of 20oC/min, equilibrated at 40oC, and then ramped up to 150oC for TPV, and

169

150oC for HPMCAS and PVPVA, using a heating rate of 20oC/min. For HPMC only one thermal

170

cycle was used. From the resulting thermograms, Tgs were determined as the midpoint of the glass

171

transition in the heat flow curve of the second heating ramp. All samples were prepared in

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triplicate.

173 174

Nano TA measurements

175

Localized thermal analysis was conducted using the nanoIR2TM AFM-IR instrument

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(Anasys Instruments, Inc., Santa Barbara, CA) in nanoTA mode. A nanoTA ramp is obtained by

177

heating the probe linearly with time while the extent of cantilever bending is recorded. When a



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thermal event occurs, the sample surface becomes softened and then the AFM tip penetrates into

179

the sample (Figure S2, supporting information). The local maximum in the temperature ramp is

180

typically defined as the onset of the thermal event 20.

181

ThermaleverTM probes (Model: EXP-AN2-300, Anasys Instruments Inc., Santa Barbara,

182

CA) were used. Briefly, the AFM tip was heated linearly with time, and the bending of the probe

183

was recorded. A thermal event is defined as penetration of the probe into the surface of the sample

184

due to sample softening 20. Prior to data collection, a calibration curve was made by measuring the

185

glass transition temperature of polymeric calibration samples polycaprolactone (55oC),

186

polyethylene (116oC), and polyethylene terephthalate (235oC). A plot of deflection versus heating

187

voltage was generated by the software. The calibration curve was obtained by fitting the three

188

points using a quadratic fit. Voltage was then converted to temperature after temperature

189

calibration. After calibration, the telaprevir and polymer films were analyzed. An AFM

190

topographical image was acquired prior to nanoTA ramps to locate the discrete and continuous

191

domains. The probe was heated at a rate of 5oC/s. A deflection decrease of 0.15 V within 20 ms

192

was set as the first trigger abort, normally indicating a thermal transition of the sample. A

193

resistance decrease value of 0.15 V within 20 ms was set as the second trigger abort. This was

194

used to prevent probe damage caused by passing the resistance turnaround point of the probe. The

195

first derivative of the deflection versus temperature curve was taken, and the intersection at y=0

196

was calculated as the softening point.

197 198

Lorentz Contact Resonance (LCR) measurements

199

Lorentz contact resonance (LCR) AFM is a type of contact resonance AFM. In contact

200

resonance AFM, information about the viscoelastic properties of a sample in contact with an AFM


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probe can be evaluated at the nanoscale by measuring the contact stiffness between the probe and

202

the sample

203

resonance modes of the cantilever are monitored

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oscillation in a cantilever including piezoelectric, electrostatic, photothermal, thermomechanical

205

and magnetic approaches

206

using a U-shaped AFM probe that conducts oscillating electricity in a magnetic field to generate

207

an oscillating force

208

the mechanical properties of the material in contact with the AFM tip. By recording the oscillation

209

amplitude as a function of position at a certain frequency, surface images that reflect differences in

210

the relative stiffness of each component can be obtained. The advantage of LCR over other contact

211

resonance techniques, such as piezoelectric and ultrasonic, is that the nanomechanical spectra

212

generated by LCR is free from parasitic peaks generated by the piezo

213

differentiates different regions on the sample surface based on the stiffness of the sample, rather

214

than phase, which could be affected by fiction, viscosity, or other adhesive forces 22, 25. In addition,

215

by simply modulating the alternating current, a single AFM cantilever can be used to analyze

216

samples with a wide range of stiffness 22, 24, 25.

21, 22

. The AFM cantilever is oscillated and the amplitude and frequency of the

26

24, 25

23

. There are several methods for generating an

. The Lorentz contact resonance (LCR) technology is achieved by

. The amplitudes and peak frequencies of the oscillation are determined by

22, 24, 27, 28

. Also, it

217

ThermaleverTM probes (Model: EXP-AN2-300, Anasys Instruments Inc., Santa Barbara,

218

CA) were used together with a small LCR drive magnet to conduct LCR sweeps and collect LCR

219

images. Prior to LCR sweeps, an AFM height scan was performed. LCR sweeps were then

220

conducted by placing the AFM cantilever on areas of interest to collect nanomechanical spectra. A

221

drive strength of 20% was used for LCR sweeps. The starting and ending values were set to 1 kHz

222

and 1000 kHz, respectively, with a scan rate of 100 kHz/s. The data collection rate was set to 200

223

pt/s. In addition to nanomechanical spectra collection, LCR images could be obtained by driving



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224

the AFM cantilever at one of the contact resonance modes. For a heterogeneous sample, a contact

225

frequency that shows differences among mechanical spectra obtained from different regions on the

226

sample is used. If the contact frequency between the AFM probe and the sample is the same as the

227

value at which the probe is driven, bright areas will be visible in LCR amplitude images. If

228

multiple regions on the sample have the same contact frequency, the LCR amplitude image will

229

show contrast based on amplitude differences at this frequency value across the scan area. An

230

LCR drive strength value of 50% was used to collect LCR images. The x and y resolution values

231

were set at 256 and 256pt, with a scan rate of 0.3 Hz. The image acquisition time was

232

approximately 14 minutes.

233 234

Fluorescence microscopy

235

Drug-polymer stock solutions were prepared at different drug-polymer ratios in methanol:

236

dichloromethane (1:1, v/v) mixed solvents at 5% solid contents. Pyrene was added as a

237

fluorescence probe to a final concentration of 0.01% (w/v). 100µL of the stock solution was spin-

238

coated onto a quartz slide and then dried overnight in a vacuum oven. Fluorescence images were

239

obtained using an Olympus BX-51 fluorescence microscope (NY, USA). A filter was used to

240

provide excitation from 330-380nm and a separate filter was used to collect the emission from

241

420nm and above.

242 243

Results

244

Topographical imaging


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245

The AFM height images for spin coated films of the pure compounds are shown in Figure

246

2A. The topographical features of telaprevir-polymer systems at different drug-to-polymer ratios

247

are shown in Figure 3.

248

The AFM images for both pure telaprevir and pure HPMC showed a smooth surface with

249

very few topographical features. For telaprevir-HPMC systems, discrete domains were observed at

250

10% drug loading. As the drug loading increased, these height features became larger. As the drug

251

loading increased from 40% to 50%, the domains present in the height image became more

252

irregular in shape and less uniform in size. At a 50% drug loading, the domain height is about

253

30nm. Based on the change in domain size with drug loading, the discrete domains are most

254

likely telaprevir-rich, while the continuous domains are HPMC-rich.

255

The height image for the pure HPMCAS film showed some variations in topography

256

suggesting that the film formation was uneven. For the telaprevir-HPMCAS films, similar

257

variations in height were also observed at drug loadings below 40%. The areas of the film showing

258

height variation are irregular in shape. When the drug loading level increases to 40%, very small,

259

regularly shaped, circular domains of about 120nm in diameter and 50nm in height are formed.

260

These features become more obvious when drug loading reaches 50%. From these images, it

261

appears that phase separation may have occurred when the drug loading exceeds 40%, although

262

additional investigations are need to confirm this.

263

For TPV-PVPVA films, discrete domains were also observed at a 10% drug loading. The

264

distance between these domains decreases with increasing drug loading. These discrete domains

265

grow taller and hence the sample surface becomes rougher. To avoid surface damage, tapping

266

mode was used instead of contact mode at 30-50% drug loading levels. The diameter of these

267

discrete domains increases slightly from around 280nm to 380nm as drug loading increases from



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268

10% to 50% and their height increases from around 20nm to 100nm. Therefore, these discrete

269

domains are probably telaprevir-rich, while the continuous phase is PVPVA-rich.

270

Although AFM imaging allows detailed surface topographical features to be evaluated, it is

271

hard to conclusively determine if these systems are phase separated, for instance, consider the

272

TPV-HPMCAS system at 50% DL which shows very small domains, or to elucidate the chemical

273

composition of each domain. AFM-IR spectroscopy, however, can provide information on the

274

chemical composition of specific topographical features and has been used previously to evaluate

275

drug-polymer miscibility

276

behavior of these drug-polymer thin film systems.

29

. It is therefore a powerful tool to further explore the miscibility

277 278

AFM-IR spectroscopy

279

The reference IR spectra from 1200-1800 cm-1 for pure telaprevir and the polymers are

280

shown in Figure 2B and C. HPMC has very weak AFM-IR signal in this region and therefore has a

281

low signal to noise ratio. In general, the peaks at wavenumbers above 1600cm-1 are less intense in

282

the AFM-IR spectra as compared to in the bulk IR spectra. Such discrepancies might be caused by

283

the differences in hardware and laser settings

284

AFM-IR spectra are consistent with those shown in bulk IR spectra. From both the bulk and AFM-

285

IR spectra, it can be seen that the peak at 1525cm-1 is only present in telaprevir, and is absent in

286

the spectra of all of the polymers. Due to the highly complex molecular structure of telaprevir,

287

attempts at theoretical calculations of the IR spectrum of telaprevir were not successful. Based on

288

the peak position, the peak at 1525 cm-1 may be a ring stretch for the pyrazine ring, which is

289

known to give rise to an infrared absorption peak in this region

290

determine the absence or presence of telaprevir in various sample regions evaluated in the AFM-

29

. However, the peak positions observed in the


30

. This peak was selected to

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291

IR experiments. As the intensity of the localized IR signal is strongly affected by sample thickness

292

31

, i.e. topography, peak ratios instead of peak intensities are used for comparison.

293

Local IR spectra obtained from a 60:40 (w:w) TPV-HPMC system are shown in Figure 4A.

294

For the continuous phase, the peak at 1450 cm-1 is of comparable intensity to the peak at 1525 cm-

295

1

296

composition of the discrete domains which show up as higher regions in the height image are

297

different from the continuous phase, with the IR data indicating that the dispersed phase is

298

telaprevir-rich, and the continuous phase is HPMC-rich. The characteristic IR peaks for telaprevir

299

and HPMC are present in both the continuous and dispersed phases. This can be caused either

300

because of partial miscibility between telaprevir and HPMC, or because there is a certain degree of

301

spectral mixing vertically through the thickness of the sample or laterally between the two

302

domains at a distance below the spatial resolution of the technique, or some combination of these

303

two factors 29.

, whereas it is considerably reduced in intensity in the dispersed phase. Therefore, the chemical

304

For TPV-HPMCAS systems, the height features observed at 50% drug loading level are

305

small and crowded, leaving the continuous phase with very limited space. The maximum distance

306

among the discrete domains are 21nm and 25nm for TPV-HPMCAS with 40% and 50% DL,

307

respectively. To date, the best lateral resolution obtained with the AFM-IR technique is around

308

20nm

309

resolution of AFM-IR is affected by film thickness and material thermomechanical properties 31, 33,

310

34

311

analytically challenging for the AFM-IR instrument to detect signals from each region without

312

interference. Therefore, the localized IR spectra were collected at an increased drug loading level

313

of 80%. The normalized local IR spectra for the 80:20 (w:w) TPV-HPMCAS system are shown in

32

. However, for systems with very small domain sizes, it may still be challenging, as the

. A typical spatial resolution of AFM-IR was reported to be around 100nm


31, 33, 35, 36

. Thus it is


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Page 16 of 46

314

Figure 4B. The peaks at 1525 cm-1 have similar intensities in both the continuous and dispersed

315

phases. However, there is a clear increase in the intensity for the peak at 1236 cm-1 in the spectra

316

obtained from continuous phase, which is a HPMCAS specific peak (see Figure 2). Therefore, it is

317

apparent that the height features observed in this system are most likely due to phase separation

318

and the discrete domains appear to be drug-rich based on the IR spectra. Phase separation

319

appeared to occur when the drug loading exceeded 30%. Because the formation of two phases was

320

not observed until relatively high drug loadings, this system has a higher extent of miscibility

321

between the two components relative to TPV and HPMC. This makes it harder to chemically

322

discriminate the two phases since each phase will contain a relatively high amount of telaprevir. In

323

addition, it was observed that the miscibility of TPV and HPMCAS seemed to be somewhat

324

dependent on environmental moisture, especially for samples around 20-50% drug loading which

325

showed some day-to-day variations in microstructure. Unfortunately, it is not currently possible to

326

control RH during sample measurement, although it is known that water can promote phase

327

separation

328

clearly an interesting observation that warrants further study.

15, 37, 38

. Given that amorphous formulations contain some level of moisture, this is

329

For the TPV-PVPVA systems, localized IR spectra obtained from a 50:50 (w/w) system

330

are shown in Figure 4C. In the continuous phase, the intensity of the peak at 1244cm-1 (a PVPVA

331

specific peak) is greater than the telaprevir peak at 1525cm-1; whereas in the dispersed phase it is

332

weaker relative to the TPV peak at 1525cm-1. Similarly, spectral mixing is also observed in this

333

system. Preliminary analyses of the results suggest that the dispersed phase is telaprevir-rich, with

334

the continuous phase being rich in PVPVA.

335

To further elucidate the evolution of chemical composition in these systems with

336

increasing telaprevir concentration, the localized IR spectra for the dispersed phase for TPV-


Page 17 of 46

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337

polymer systems were collected with increasing drug loading levels as shown in Figure 5. The

338

continuous phase was not investigated for these systems, as there was limited space to place the

339

AFM tip in between the discrete regions. The peak intensity at 1525 cm-1 was observed to increase

340

with increasing drug-loading levels, and, compared to the telaprevir peak, the intensities of the

341

polymer-characteristic peaks decreased. This might suggest that the telaprevir concentration

342

increased in the dispersed phase as drug loading increased. However, from a theoretical

343

perspective, this is not what would be expected, since phase separation should lead to two phases

344

of constant composition, whereby changing the drug loading within the region of the phase

345

diagram where there is a miscibility gap should only change the amount of each phase, rather the

346

composition of each phase. One explanation might be that the system is not in equilibrium and is

347

kinetically trapped. A more likely explanation is that these observations arise from limitations of

348

the analytical method in terms of being able to obtain spectra exclusively from each phase, without

349

contributions from the other phase, leading to spectral mixing; the extent of spectral mixing will

350

depend on the size and thickness of the domains. It can be noted that as the drug loading

351

increases, the size of the domains increases, both laterally and in the Z-direction. Consequently,

352

the higher apparent drug concentration in the disperse phase with increasing drug loading may be

353

a result of the increasing size of the drug-rich domains, and hence a lower spectral contribution

354

from the dispersed phase.

355 356

AFM-IR imaging

357

AFM-IR imaging, was performed based on select films. Peaks at 1525 and 1457 cm-1 for

358

TPV-HPMC systems, 1525 and 1740 cm-1 for TPV-HPMCAS systems, and 1525 and 1676 cm-1

359

for TPV-PVPVA systems were selected for IR imaging purposes. Drug loadings of 60%, 80%,



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Page 18 of 46

360

and 30% were chosen for TPV-HPMC, TPV-HPMCAS, and TPV-PVPVA systems respectively as

361

these films contained domains of the appropriate size to generate significant IR signal contrast and

362

enable imaging of the continuous phase. The topographical image, IR image at 1525 cm-1

363

(telaprevir peak), ratio image of the two images obtained at the aforementioned two wavenumbers,

364

and the contact resonant frequency image obtained at 1525 cm-1 are shown in Figure 6.

365

For all systems, IR images obtained at 1525 cm-1 agree very well with the topographical

366

images whereby higher telaprevir signal corresponds to the discrete domains that are higher in

367

height. IR images at 1740 cm-1 for TPV-HPMCAS system, and that at 1676 cm-1 for TPV-PVPVA

368

system (polymer peaks), show similar patterns, i.e. higher absorbance for the drug-rich domains,

369

and lower absorbance in the polymer-rich domains. The AFM-IR signal intensity is linearly

370

dependent on the thickness of the absorbing material, which in these samples is determined by the

371

sample topography, in addition to other material properties such as the thermal expansion

372

coefficients of each component

373

independent leading to changes in relative peak intensities, as shown in the localized IR spectra

374

(Figure 4 and 5). Therefore, IR ratio images, instead of measurements at a single wavenumber, can

375

be used to reduce the influence of these factors and should be considered for miscibility evaluation

376

purposes. The ratio images for all samples shown in Figure 6 correspond very well with their

377

topographical images, confirming these systems are phase separated. For these images, which are

378

based on the ratio of a polymer peak/a drug peak, a higher intensity is seen for the continuous

379

phase relative to the discrete phase, confirming that it is polymer-rich.

29, 31

. However, these effects should be relatively wavelength

380

Because the cantilever acts like a spring-damper system where the tip resonant frequency

381

changes with variations in surface stiffness, frequency maps of heterogeneous samples can be

382

obtained by measuring the contact resonant frequency of the cantilever as a function of position on


Page 19 of 46

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383

the sample 19. The cantilever oscillation is produced by the thermal pulse in the sample that results

384

from the IR radiation

385

AFM cantilever and the mechanical properties of the material the AFM probe is in contact with.

386

As the stiffness of the sample increases, the contact resonance will increase

387

images, shown in the lower panel of Figure 6, correspond well with the topographical features. For

388

TPV-HPMC and TPV-HPMCAS systems, the drug-rich phase (red colored domains) are stiffer

389

than the surrounding polymer (blue-colored regions in the frequency maps). For TPV-PVPVA

390

systems, the continuous phase is mostly uniform and lower in frequency, relative to the higher

391

frequency of the discrete domains, suggesting the polymer-rich phase is slightly stiffer than the

392

drug-rich domains.

19

. The frequency of this signal is a function of both the properties of the

19

. The frequency

393 394

Nano Thermal analysis

395

The Tgs of telaprevir, HPMC, HPMCAS, and PVPVA as measured using DSC are

396

summarized in Table 1. The Tg of telaprevir was determined to be 103oC, consistent with the

397

previously reported value of 105oC 4. It can be seen that the Tgs of telaprevir and PVPVA are very

398

similar, and hence bulk thermal measurements of Tg using a method such as DSC cannot be

399

readily employed to detect phase separation in such systems. NanoTA analysis, on the other hand,

400

can provide information about the thermal properties of individual domains. This is potentially

401

useful to provide information about the chemical composition and physical state of materials in

402

each domain in a phase separated system. The glass transition temperatures of the pure

403

components measured from spin coated films with nanoTA are shown in Table 2. For pure

404

amorphous telaprevir, the glass transition temperature measured by DSC is consistent with the

405

softening value measured by nanoTA. However, for pure polymers, there are some discrepancies



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Page 20 of 46

406

between the values measured by these two techniques. The softening temperatures measured by

407

nanoTA are around 20-60oC higher than those measured by DSC.

408

The softening temperatures of individual domains in the phase separated systems were also

409

measured using nanoTA. A 60:40 (w/w) TPV-HPMC system, a 80:20 (w/w) TPV-HPMCAS

410

system, and a 30:70 (w/w) TPV-PVPVA system were used for nanoscale thermal analysis. The

411

results of local thermal analysis are shown in Figure 7. For the TPV-HPMC system, the softening

412

thermal event for the discrete regions occurred at around 130oC, consistent a thermal event

413

associated with a telaprevir-rich phase. It occurred at a temperature a little above the softening

414

temperature observed for pure telaprevir by nanoTA, possibly due to the presence of some HPMC

415

in this phase. Furthermore, the observation of a softening temperature in this temperature range

416

confirms that telaprevir is present in the amorphous state in the thin films and hence phase

417

separation is not due to crystallization; the melting point of telaprevir was measured by DSC to be

418

240°C. The thermal event observed in the continuous phase occurred at around 156oC, consistent

419

with the glass transition of an HPMC-rich phase. Similarly, the softening temperature for the

420

continuous phase was below that for pure HPMC, most likely due to the plasticization effect of

421

amorphous TPV. These results confirmed the partial miscibility of TPV and HPMC in these phase

422

separated domains. For the TPV-HPMCAS system, the thermal events observed for the

423

continuous and dispersed phases occurred at around 124oC and 143oC, respectively. This is

424

consistent with glass transition events for TPV- and HPMCAS-rich phases respectively. The

425

nanoTA thermograms obtained from the continuous and dispersed phases in the TPV-PVPVA

426

system are shown in Figure 7C. Here it is apparent that the thermal events for both the continuous

427

and dispersed phases occurred over a similar temperature range. This is readily explainable

428

because the glass transition temperatures for PVPVA and TPV are very similar (Table 1). The


Page 21 of 46

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429

softening temperature for the continuous phase is higher than that of pure PVPVA. This may be

430

caused by intermolecular interactions between the two components 39.

431 432 433

Lorentz Contact Resonance (LCR) imaging To further explore the mechanical properties of each phase, Lorentz contact resonance 26

434

measurements were performed

. The mechanical spectra of pure telaprevir and polymers are

435

shown in Figure 8. Higher frequencies correspond to stiffer materials 40. The first and largest peak

436

represents the first flexural resonance mode, which is normally chosen for LCR imaging purposes

437

because of its high sensitivity and stiffness selectivity. It can be seen that the peaks for both

438

HPMC and HPMCAS appear at lower resonance frequency ranges compared to the telaprevir peak

439

(Figure 8B), confirming that telaprevir is slightly stiffer than HPMC and HPMCAS. It can also be

440

seen that HPMC is slightly softer than HPMCAS. Therefore, by scanning at these peak

441

frequencies, LCR images highlighting drug-rich and polymer-rich can be potentially obtained.

442

However, in this frequency range, PVPVA and TPV have virtually identical peak resonance

443

frequencies. To help differentiate these two components, a higher mode of the cantilever was

444

selected, and the second largest peak at around 710 kHz was used for LCR imaging for TPV-

445

PVPVA systems. The PVPVA peak appears at a higher frequency value compared to that of

446

telaprevir (Figure 8C), confirming PVPVA is slightly stiffer than TPV.

447

LCR images were then obtained at the selected contact resonant frequencies identified

448

from the mechanical spectra, where the cantilever oscillation amplitude differs depending on

449

material stiffness. The actual peak frequency used for the LCR imaging may vary slightly

450

compared to those shown in the mechanical spectra of the pure components shown in Figure 8.

451

This is due to the fact that when collecting a LCR image, the probe is translating across the



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Page 22 of 46

452

sample, and the friction between the sample and probe induces a torque on the cantilever shifting

453

the resonant frequencies. Also, the drug-polymer systems may be partially miscible and the

454

presence of a second component in each domain would be expected to alter the mechanical

455

properties somewhat compared to those of the pure components. Therefore, the peak frequency

456

used was optimized for each system to provide maximum contrast. The LCR images (Figure 9)

457

showed good contrast at the two frequencies selected for TPV and the polymers and clearly

458

indicate the presence of two phases with different mechanical properties. For TPV-HPMC and

459

TPV-HPMCAS systems, for the LCR images obtained at 128kHz, the discrete domains give rise

460

to a stronger signal (more yellow color) than the continuous phase, indicating the presence of

461

telaprevir, which has a peak at this frequency. In contrast, the continuous phase gives rise to a

462

stronger signal at 123 kHz for TPV-HPMC and 124kHz for TPV-HPMCAS systems, suggesting

463

that these areas are have a high concentration of polymer. These results confirmed that the

464

polymer-rich regions are softer as compared to the drug-rich regions. Interestingly, for the TPV-

465

HPMC system, some of the small discrete regions highlighted in red circles in Figure 9A appear to

466

be polymer-rich, rather than drug-rich based on the mechanical images. This can be confirmed

467

from the IR images shown in Figure 6A where is it is apparent that these small discrete domains

468

do not show strong IR absorbance at 1525cm-1.

469

For the TPV-PVPVA system, the PVPVA-rich (continuous phase) phase was indicated in

470

the LCR images obtained at 129kHz. The contrast obtained at this frequency is mostly caused by

471

differences in resonance peak intensity between the two materials whereby the peak height for

472

PVPVA is significantly higher than that for telaprevir (Figure 8B). A higher frequency value of

473

710 kHz, where the drug and the polymer showed differences in mechanical properties, was

474

selected to identify the telaprevir-rich regions. Unlike AFM-IR imaging where the resolution is


Page 23 of 46

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475

often limited by the properties of the sample (sample thickness, thermal diffusivity, etc), the

476

spatial resolution of LCR imaging is restricted by the tip radius of the cantilever used, resulting in

477

greatly enhanced resolution between the two phases compared to AFM-IR imaging.

478 479

Fluorescence imaging

480

To confirm phase separation in these telaprevir-polymer systems using a technique other

481

than AFM, fluorescence images were taken for these systems. Fluorescence imaging has been

482

used recently to identify phase separation in drug-polymer mixtures, and is based on the

483

preferential localization of a fluorescent additive in one of the phases

484

fluorescence microscopy images of films containing pyrene are shown in Figure 10. In a

485

chemically homogenous film such as HPMCAS (Figure 10A), it can be seen that pyrene

486

molecules are evenly distributed and no heterogeneity in fluorescence intensity was observed. In a

487

phase separated system, pyrene tends to accumulate in the more hydrophobic phase (the drug-rich

488

phase), therefore resulting in higher fluorescent emission in these regions

489

lower drug loadings, the domain sizes are below the diffraction limit of optical microscopy.

490

Therefore, higher drug loadings systems were chosen for fluorescence imaging. As shown in

491

Figure 10B-D, the dispersed phase showed higher fluorescence intensity than the continuous

492

phase, providing further confirmation that the dispersed phase is drug-rich.

41

41

. Representative

. For systems with

493 494

Discussion

495

As the utilization of amorphous solid dispersions as a means to overcome solubility limited

496

oral bioavailability grows, characterization of drug polymer miscibility becomes an increasingly

497

important topic. Various computational and experimental methods to explore drug-polymer



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16, 42-44

Page 24 of 46

498

miscibility have been employed

499

most common methods that has been used to evaluate miscibility, but suffers from a number of

500

limitations, motivating the implementation of alternative approaches

501

characterization technique should provide information about both domain size and composition in

502

samples showing poor miscibility. Composition information is important because the lower the

503

polymer content in the drug-rich phase, then the greater the likelihood of product failure via

504

crystallization during storage; if the majority of the polymer is present in a separate phase, then it

505

will be unable to inhibit crystallization as effectively as compared to a miscible blend. Domain

506

size is important since this is likely to impact the performance of the ASD during dissolution.

507

From the limited number of studies that have explored this feature, it appears that the domain size

508

of phase-separated regions is typically less than a micron14, 16, 29, 41. The small domain size of

509

phase separated regions limits the range of available analytical techniques, precluding those with

510

low spatial resolution limits, which encompasses many routine pharmaceutical characterization

511

tools. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) has emerged as a valuable

512

tool for miscibility characterization, but typically requires long analysis times, of the order of

513

several hours

514

allow interrogation of several types of material properties at sub-micron resolution, also offer

515

promise in the area of miscibility evaluation29, 41, 48. These AFM based techniques include AFM-

516

IR, nano-TA and nano-mechanical analysis. Herein, we have compared the type of information

517

that can be harnessed from the application of these techniques for miscibility evaluation of

518

telaprevir-polymer blends. This is a challenging system to evaluate since the drug has a very high

519

glass transition temperature, which is similar to that of the polymers investigated. This

520

immediately precludes the use of conventional thermal methods such as DSC. Furthermore, the

16, 46, 47

. Differential scanning calorimetry is probably one of the

10, 11, 45

. Ideally, the

. Recent developments in atomic force microscopy-based techniques that


Page 25 of 46

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521

TPV-HPMC and TPV-HPMCAS systems have very small domain sizes (below 50nm in a 10:90

522

(w/w) TPV-HPMC system and around 100-200nm in the other systems), which requires the use of

523

high-resolution imaging techniques. This limits the application of standard vibrational

524

spectroscopic imaging techniques which are restricted in terms of spatial resolution to a few

525

hundred nanometers by the diffraction limit of light. Furthermore, it is well known that differences

526

in surface features, which are readily discernable with standard AFM imaging techniques, do not

527

always correlate to differences in composition, therefore orthogonal characterization techniques

528

are needed in order to fully characterize the system properties 49-52.

529

AFM-IR has been shown previously to provide detailed chemical information for different

530

domains in drug-polymer mixtures 18, 29, 41, 48. In principle, the chemical composition of each phase

531

can be evaluated in a phase-separated system. However, the spatial resolution of AFM-IR can be

532

limited by various factors. In this study, an IR imaging attempt for a 50:50 (w/w) TPV-HPMCAS

533

system was not successful, possibly due to the small domain size of this system (122nm wide and

534

13nm high), or because of low spectral contrast between the two phases, or come combination of

535

the two issues. The low spectral contrast in this instance is due to the relatively high miscibility of

536

the telaprevir-HPMCAS system; the system only showed phase separation when the drug loading

537

exceeded 30- 40%. Therefore, when phase separation occurs, the composition difference between

538

the two phases is much smaller than for systems that show phase separation at very low drug

539

loadings and have a larger miscibility gap. Consequently, the spectra from each domain are similar

540

since both phases contain both TPV and HPMCAS. The small domain size also can be

541

problematic because it becomes difficult to obtain the IR response exclusively from the relevant

542

domain. This is because the IR radiation may penetrate through the domain such that the

543

underlying material contributes to the observed spectrum. In this study, localized IR spectra



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Page 26 of 46

544

obtained from the dispersed and continuous phases for a 50:50 (w/w) TPV-HPMCAS system were

545

identical (Figure S3, supporting information). Therefore, no chemical discrimination could be

546

achieved for the 50:50 (w/w) TPV-system via nanoIR. In contrast, the TPV-PVPVA film showed

547

signs of discrete domain formation at much lower drug loadings based on the topographical

548

images (Figure 3C), and nanoIR could readily distinguish the drug-rich and polymer-rich phases at

549

a 30% drug loading (Figure 6).

550

Nano-TA analysis can reveal the physical state of individual domains on the surface of the

551

sample. Not only can it be used to confirm that these telaprevir-polymer systems are phase-

552

separated based on different localized thermal softening behavior, but it also provides

553

confirmation that telapreivr exists in an amorphous form in these solid dispersion films over the

554

time course of this study. However, its applicability is limited for samples with similar Tgs.

555

Consequently, for the systems under evaluation in this study, this approach provided the most

556

useful data for the dispersions made with the higher Tg cellulose derivatives.

557

Nano scale mechanical spectroscopy and LCR imaging are independent of material

558

absorptivity and sample thickness, and therefore can potentially overcome some of the limitations

559

that were observed with AFM-IR for these systems. Indeed, we found that greatly enhanced spatial

560

resolution could be achieved with LCR imaging. This method is thus especially beneficial for

561

systems with very small domain sizes and weak IR absorptivity. The smallest height feature that

562

was discriminated with LCR imaging (highlighted in blue circles) had a diameter of around 98nm

563

(Figure 9A). LCR images also showed good contrast for the 50:50 (w/w) TPV-HPMCAS system

564

(data not shown) where the average domain size was 122nm and the maximum space among

565

discrete domains is 25nm; no contrast between phases could be achieved with AFM-IR for this

566

system.


Page 27 of 46

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567

It is important to understand that these nanoscale techniques sample different volumes of

568

material when making the measurement, impacting the spatial resolution and the depth sensitivity.

569

The nanomechanical measurements, such as LCR, are limited in spatial resolution by the contact

570

area between the probe and sample. The contact area depends on the tip radius used, the normal

571

force applied and the stiffness of the sample, so is somewhat variable, but is typically in the 10 nm

572

range if the force is minimized. The depth sensitivity is similar to the contact area, so this is

573

strictly a surface sensitive technique. The nanoTA technique is similar to the nanomechanical

574

technique, but typically higher normal forces are applied between the tip and sample. In addition

575

once the sample undergoes a thermal transition, it will typically soften significantly leading to a

576

larger penetration into the sample. This will result in a typical spatial resolution of 100 nm and a

577

depth sensitivity of approximately half that. The AFM-IR technique works by measuring the

578

oscillation of the cantilever caused by the rapid expansion of the sample due to absorption of the

579

IR illumination. The volume of material which expands depends on the thickness of the absorbing

580

material, the absorption coefficient of the material, and any mechanical constraints which limit the

581

expansion of the material. Typically, to achieve the highest spatial resolution, the thickness of the

582

absorbing material needs to be thin, on the order of a few 100 nanometers. This will lead to a

583

spatial resolution of ~100 nm. The AFM-IR signal is an integration of the absorption of the IR

584

light through the thickness of the material, and so is not limited to the surface of the material,

585

meaning that if there is material variation through the thickness of the sample, the resultant AFM-

586

IR signal will be a mix of these components.

587

Nanoscale IR, TA, and LCR analysis are clearly powerful tools in miscibility evaluation,

588

particularly when used in concert. These AFM-based techniques can be used to determine the

589

miscibility behavior of systems that are ambiguous and analytically challenging, and obtain



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Page 28 of 46

590

detailed information on sample chemical composition and physical properties. Combined with

591

AFM imaging, these techniques enable miscibility to be evaluated at high spatial resolution,

592

providing information about both domain size and chemical composition of each of the phases.

593

The results of this study indicate that telaprevir and HPMCAS are phase separated at drug

594

loading levels above 30% when prepared as thin films prepared by spin coating under ambient

595

conditions. Furthermore, the TPV-HPMCAS system appears to be the most miscible of

596

dispersions tested with ASDs formed with PVPVA or HPMC showing signs of phase separation at

597

lower drug loadings. Interestingly, the commercial formulation of telaprevir (Incivek®) is an

598

amorphous solid dispersion product formulated with HPMCAS, and produced by spray drying.

599

Clearly it would be of interest to further investigate the impact of excipients and processing

600

conditions on the microstructure and miscibility of TPV-HPMCAS, applying some of the

601

techniques that have been described herein. Advanced sample preparation techniques, such as

602

embedding and microtoming, are likely to be required to enable the evaluation of spray dried

603

particles with these methods.

604 605

Conclusions

606

The miscibility of telaprevir with three different polymers was evaluated using nanoscale

607

infrared spectroscopy, thermal analysis, and Lorentz contact resonance measurements. It was

608

challenging to characterize miscibility for some of these systems due to the similarity in Tgs of the

609

components as well as the small domain sizes of the phase-separated regions. By combining the

610

chemical composition information obtained from nanoscale infrared spectroscopy, the softening

611

behavior as evaluated from nano thermal analysis, mechanical analysis of the samples using

612

Lorentz contact resonance measurements, with standard AFM imaging, it was possible to


Page 29 of 46

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613

characterize the microstructure as a function of polymer type and amount and to determine that

614

phase separation had occurred. The drug-rich phase was found to form discrete domains of various

615

sizes depending on the system, ranging from below 50 nm to a few hundred nanometers, while the

616

continuous phase was polymer-rich. The nanoscale characterization techniques provided detailed

617

information about surface features, chemical composition, and physical properties. Such

618

information is essential to better understand relationships between microstructure and product

619

performance.

620 621

Acknowledgements

622

The authors would like to thank the National Science Foundation through grant number

623

IIP-1152308, the National Institutes of Health through grant numbers R41 GM100657-01A1 and

624

R42 GM100657-03, and the United States Food and Drug Administration under Grant Award

625

1U01FD005259-01 for financial support. We gratefully acknowledge Kevin Kjoller, Michael Lo,

626

Caitlin Schram, and Aaron Harrison for technical training and helpful discussions.

627 628

Abbreviations

629

AFM, atomic force microscopy; API, active pharmaceutical ingredients; ASD, amorphous

630

solid

dispersion;

HPMC,

hydroxypropyl

methylcellulose;

631

methylcellulose acetate succinate; IR, infrared; LCR, Lorentz contact resonance; TA, thermal

632

analysis; PTIR, photothermal-induced resonance; PVPVA, polyvinylpyrrolidone/vinyl acetate;

633

TPV, telaprevir.

634 635

Associated content


HPMCAS,

hydroxypropyl


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

636

Page 30 of 46

Supporting information

637

A representative optimization map showing a distinct IR hotspot (Figure S1), sample

638

topography after nanoTA ramps (Figure S2), and normalized localized IR spectra for a 5:5 (w/w)

639

TPV-HPMCAS system (n=5) A) original B) overlaid (Figure S3) are provided as supporting

640

information. This material is available free of charge via the Internet at http://pubs.acs.org.

641


Page 31 of 46

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642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685


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731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792

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793 794


Page 34 of 46

Page 35 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

795


Figures

O NH O O

O H N

N N H

OR

OR NH H

O

N

O O

O N H

OR

n R=H or CH3 or CH2CH(OH)CH3

telaprevir

CH 2OR O OR

HPMC

OR O

O OR

CH 3

OR O CH 2OR

C n

R=H, CH3, CH2CH(OH)CH3, COCH3, COCH2CH2COOH, CH2CH(OCOCH3)CH3, CH2CH(OCOCH2CH2COOH)CH3

796 797 798

N CH

O CH 2

n

O

O

CH

CH 2

PVPVA

HPMCAS

Figure 1 Molecular structures of telaprevir and polymers


m


A

B

C

Absorbance

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 36 of 46

1800

799 800 801 802 803

1600

1400

1200

Wavenumber (cm-1)

1800

1600

1400

1200

Wavenumber (cm-1)

Figure 2 Reference mid-IR spectra and topographical images (1200-1800 cm-1, normalized) of telaprevir and polymers (From top to bottom: TPV, HPMC, HPMCAS, PVPVA). (A) topographical images (B) bulk transmission spectra and (C) localized IR spectra obtained from spin coated films of pure substances (n=9)


Page 37 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

804 805 806


A

B

C

Figure 3 Topographical images of TPV-polymer systems at different drug loadings (From top to bottom: 1:9, 2:8, 3:7, 4:6, and 5:5 drug to polymer ratio) (A) TPV-HPMC (B) TPV-HPMCAS (C) TPV-PVPVA



807 A

Absorbance

continuous

dispersed

1800

1600

1400

1200

Wavenumber (cm-1)

808 B

Absorbance

continuous

dispersed

1800

1600

Wavenumber

809

1400

1200

(cm-1)

C

continuous

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 46

dispersed

1800

810 811 812 813

1600

Wavenumber

1400

1200

(cm-1)

Figure 4 Topographical images and normalized localized IR spectra TPV-polymer systems (A) 6:4 (w/w) TPV-HPMC system (n=4) (B) 8:2 (w/w) TPV-HPMCAS system (n=5) (C) 5:5 (w/w) TPV-PVPVA system (n=5)


Page 39 of 46

814 A

B

C

2:8

1:9

5:5

Absorbance

4:6

6:4

7:3

Absorbance

2:8 3:7

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


3:7

4:6 5:5

1800

815 816 817 818

1600

1400

Wavenumber (cm-1)

1200

8:2

1800

1600

1400

1200

Wavenumber (cm-1)

5:5

1800

1600

1400

1200

Wavenumber (cm-1)

Figure 5 Localized nanoscale mid-IR spectra of TPV-polymer systems at different drug-to-polymer ratios (n=5) (A) TPV-HPMC, dispersed phase (B) TPV-HPMCAS, dispersed phase (C) TPV-PVPVA, dispersed phase



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 46

819

820 821 822 823 824 825 826 827

A

B

C

Figure 6 Nanoscale mid-IR image of TPV-polymer systems (A) 60:40 (w/w) TPV-HPMC system(from top to bottom: topographical image, IR image at 1525 cm-1, ratio image (1457 cm1 /1525 cm-1), and frequency image) (B) 80:20 (w/w) TPV-HPMCAS system(from top to bottom: topographical image, IR image at 1525 cm-1, ratio image (1740 cm-1/1525 cm-1), and frequency image) (C) 30:70 (w/w) TPV-PVPVA system (from top to bottom: topographical image, IR image at 1525 cm-1, ratio image (1676 cm-1/1525 cm-1, and frequency image)


Page 41 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


828

829

830 831 832 833 834 835

Figure 7 NanoTA measurements of TPV-polymer systems (A) 60:40 (w/w) TPV-HPMC system (n=5 for each phase) (B) 80:20 (w/w) TPV-HPMCAS system (n=6 for each phase) (C) 30:70 (w/w) TPV-PVPVA system (n=5 for each phase)



Amplitude (V)

A

TPV HPMC HPMCAS PVPVA 0

250

500

750

1000

Frequency (kHz)

836

Amplitude (V)

B

TPV HPMC HPMCAS PVPVA 110

120

130

140

150

Frequency (kHz)

837 C

TPV PVPVA Amplitude

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

700

838 839

705

710

715

720

Frequency (kHz)

Figure 8 Reference mechanical spectra (A) original (B, C) enlarged ACS Paragon Plus Environment

Page 43 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

840 841 842 843 844 845


A

B

C

Figure 9 LCR images of TPV-polymer systems (A) 60:40 (w/w) TPV-HPMC system (from top to bottom: topographical images, LCR image at 128kHz, and LCR image at 123kHZ) (B) 80:20 (w/w) TPV-HPMCAS system(from top to bottom: topographical images, LCR image at 128kHz, and LCR image at 124kHZ) (C) 50:50 (w/w) TPV-PVPVA system(from top to bottom: topographical images, LCR image at 710kHz, and LCR image at129kHZ)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

846 847 848 849 850

Figure 10 Representative fluorescence images of TPV-polymer films containing pyrene (A) HPMCAS (B) 60:40 (w/w) TPV-HPMC (C) 80:20 (w/w) TPV-HPMCAS (D) 50:50 (w/w) TPVPVPVA


Page 44 of 46

Page 45 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

851 852 853


Table 1 Glass transition temperatures of telaprevir, HPMC, HPMCAS, and PVPVA determined by DSC Compound TPV HPMC HPMCAS PVPVA

Tg (oC) 102.7±0.4 140±5 122±1 108±1

854 855



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

856 857 858

859 860 861 862

Page 46 of 46

Table 2 Softening (glass transition) temperatures of telaprevir, HPMC, HPMCAS, and PVPVA determined by nanoTA System TPV HPMC HPMCAS PVPVA 60:40 (w/w) TPV-HPMC 80:20 (w/w) TPV-HPMCAS 30:70 (w/w) TPV-PVPVA a: pure component b: dispersed phase c: continuous phase

Tg (oC)a 102±5 209±11 151±2 132±3 NA NA NA

Tg1 (oC)b NA NA NA NA 130±2 124±14 131±5


Tg2 (oC)c NA NA NA NA 156±5 143±3 146±4

Nanoscale Infrared, Thermal, and Mechanical ... - ACS Publications

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