Frozen in Time: Kinetically Stabilized Amorphous ... - ACS Publications

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Frozen in Time: Kinetically Stabilized Amorphous Solid Dispersions of Nifedipine Stable after a Quarter Century of Storage Frank Theil, Sankaran Anantharaman, Samuel O. Kyeremateng, Holger van Lishaut, Sebastian H. Dreis-Kühne, Jörg Rosenberg, Markus Mag̈ erlein, and Gerd H. Woehrle* AbbVie Deutschland GmbH & Co. KG, 67061 Ludwigshafen, Germany S Supporting Information *

ABSTRACT: Kinetically stabilized amorphous solid dispersions are inherently metastable systems. Therefore, such systems are generally considered prone to recrystallization. In some cases, the formation of crystals will impact the bioavailability of the active pharmaceutical ingredient in these formulations. Recrystallization therefore may present a significant risk for patients as it potentially lowers the effective dose of the pharmaceutical formulation. This study indicates that such metastable formulations may indeed remain fully amorphous even after more than two decades of storage under ambient conditions. Different formulations of nifedipine stored for 25 years were compared with freshly prepared samples. A thorough physicochemical characterization including polarized light microscopy, differential scanning calorimetry, X-ray powder diffraction, and transmission Raman spectroscopy was undertaken. This in-depth characterization indicates no signs of recrystallization in the stored samples. The observations presented here prove that long-term stability of amorphous solid dispersions much beyond the typical shelf life for pharmaceutical formulations is indeed possible by kinetic stabilization alone. These findings implicate a reevaluation of the propensity to recrystallize for kinetically stabilized amorphous solid dispersions. KEYWORDS: amorphous solid dispersion, long-term stability, nifedipine, physicochemical characterization, crystallinity, recrystallization

1. INTRODUCTION Over the last decades, more and more poorly water-soluble drug substances came out of the discovery organizations of pharmaceutical companies.1 In order to enhance the bioavailability of such drugs, their solubility in biologically relevant media, e.g., aqueous solutions, needs to be increased.2 It is wellknown that the apparent solubility of an active pharmaceutical ingredient (API) can be significantly enhanced by formulation as an amorphous solid dispersion (ASD).3 The bioavailability can be drastically increased for APIs where dissolution is the rate-limiting step (biopharmaceutical classification system (BCS) II drugs) by administering the drug in form of an ASD.4 The thermodynamically stable solid phase of a substance is crystalline, rendering the amorphous phase prone to recrystallization. Naturally the API-load of an ASD has limitations, regarding its thermodynamic stability, but to lower the pill burden for the patient, high drug loads are highly desirable.5 Stabilizing high drug load ASD formulations against recrystallization is therefore a challenging task for pharmaceutical research and industry.6 With a plethora of poorly water-soluble drug molecules available, this type of pharmaceutical formulation has become more and more important over the last years.7 In an ASD typically a polymer or other carrier material (e.g., mesoporous silica) is used as stabilizer of the amorphous state © XXXX American Chemical Society

of the API. These materials decrease the onset of crystallization of the API by a reduction of molecular mobility and the disruption of molecular recognition between the single API molecules.8 In a thermodynamic picture the driving force of crystallization is decreased, and the energy barrier of the phase transition is increased.9 Since the stabilization of the thermodynamically less stable amorphous phase requires close interaction between the API and the polymer, a stabilization can only be achieved if polymer and API are mixed at a molecular level and will not be observed in the case of a mere physical mixture.3 For a mixture of high melting API and a polymer with a phase transition at a lower temperature and a given ratio of polymer to crystalline API the temperature of final crystal dissolution in the polymer is defined as the solubility temperature. This temperature rises with the mass fraction of the crystalline API. Above solubility temperature the system is thermodynamically stable; below this temperature the system is an oversaturated solution of the API in the polymer. As such, it is considered thermodynamically unstable and inherently prone to recrystallization. As a glassy solution an Received: August 25, 2016 Revised: November 3, 2016 Accepted: November 10, 2016

A

DOI: 10.1021/acs.molpharmaceut.6b00783 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

2. EXPERIMENTAL SECTION 2.1. Chemicals. Copovidone was purchased from BASF SE, Eudragit RS from Evonik Industries, and nifedipine from TEVA GmbH. KH2PO4, K3PO4·H2O, H3PO4, Na2HPO4, NaH2PO4· 2H2O and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich. Acetonitrile (HPLC grade) for HPLC measurements was purchased from J. T. Baker. All other organic solvents were of analytical grade and were purchased from Merck KGaA. 2.2. Hot Melt Extrusion (HME). Three different formulation compositions were tested that differed in the copovidone and Eudragit RS content. The drug load was 20% (w/w) in all extrudate samples. The extrudate compositions and extrusion temperatures are summarized in Table 1. For the

ASD is also characterized by the glass transition temperature Tg, characterizing the transition point between glass and supercooled liquid. The value of Tg usually decreases with increasing mass fraction of the API and the moisture content of the formulation.10 If the ASD is below the solubility temperature, hence oversaturated and thermodynamically instable, the system can still be kinetically stabilized if stored below its glass transition temperature Tg. Crystallization of the API is thermodynamically favored in this case, but the molecular motion in the ASD is almost frozen, making the phase transition reaction extremely slow.11 For an ASD to be kinetically stable over long time periods, the current rule of thumb is that the storage temperature should be low enough to slow down molecular motion, e.g., at least 50 °C below the Tg of the ASD.12 In the presented study, the stability of an ASD of the model drug nifedipine in copovidone was investigated. With a drug load of 20% (w/w) this formulation is oversaturated (Figure 1)

Table 1. Composition and Extrusion Temperatures of the Investigated Samples

Figure 1. Phase diagram of the three investigated formulations, as denoted in Table 1 (blue, A1/B1; red, A2/B2; black, A3/B3). Red and blue curves are superimposed. Filled symbols correspond to the solubility and hollow symbols to the Gordon−Taylor fit. Depending on the position relative to the solubility and Tg curve in the phase diagram, four regions can be distinguished as depicted: (i) thermodynamically stable melt, (ii) thermodynamically stable glassy solid, (iii) kinetically stable glassy solid, and (iv) the undercooled or unstable melt, respectively.

sample

nifedipine

Copovidone

Eudragit RS

T/°C

A1 B1.1 B1.2 B1.3

20% (w/w)

70% (w/w)

10% (w/w)

N/A 135 150 160

A2 B2.1 B2.2 B2.3

65% (w/w)

15% (w/w)

N/A 135 150 160

A3 B3.1 B3.2 B3.3

60% (w/w)

20% (w/w)

N/A 135 150 160

samples produced in 1991 a ZSK-30 twin-screw extruder with 30 mm screw diameter was used for manufacturing the extrudates (Werner and Pfleiderer, Stuttgart, Germany; now: Coperion). Extrusion of the new sample batch was performed by using a twin-screw 18 mm extruder (ZSK 18, Coperion) with seven barrels. A powder blend containing all ingredients of the final formulation was continuously dosed at 1 kg/h into the extruder by a loss in weight feeder system (K-tron, Coperion). The extruder screw speed was 100 rpm in all cases. Three different extruder barrel temperature settings were used for manufacturing of the three sub-batches of each of the three formulations (135, 150, and 160 °C). Please note that the formulations of the new sample batch contained 1% (w/w) SiO2 as a floating agent. 2.3. Assay Degradation. HPLC measurements were performed on a Waters 2695 separations module equipped with a Symmetry C18 (75 × 4.6 mm, 3.5 μm diameter of particle) column coupled with a Waters 2487 dual wavelength absorbance detector (Waters Corp., Milford, USA). The eluent used was a binary mixture of acetonitrile and aqueous KH2PO4buffer solution (pH = 3.0) in a linear gradient from 38% to 60% acetonitrile. Ten milligrams of the analyte were dissolved in 100 mL of the eluent by ultrasonication. Twenty-five microliters were injected for analysis. 2.4. Sample Storage. The extrudate pellets were packaged for storage in brown glass bottles closed with plastic screw caps. The samples were stored at ambient conditions without moisture and temperature control.

and therefore kinetically stabilized under ambient temperature.11 Samples of this formulation were investigated after the extraordinary long storage time of 25 years under uncontrolled ambient conditions, which is about five times longer than a typical shelf life for pharmaceutical products. The stored samples were thoroughly characterized by the pharmaceutical standard methods (assay and degradation, drug release) as well as a wide range of techniques to assess their crystalline content including polarized light microscopy (PLM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and transmission Raman spectroscopy (TRS). The results of this characterization were compared to the initial testing and to freshly prepared samples of the same high drug load formulation of nifedipine. In this encompassing study, no signs of recrystallization were found in the stored samples by means of any of the applied methods. Additionally, the drug release of the stored samples was indeed comparable to the freshly prepared extrudates of nifedipine. Those findings prove kinetically stabilized extrudates can be crystal free even over a prolonged storage period. B


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Molecular Pharmaceutics 2.5. Water Content. The water content of the samples was measured by Karl Fischer-titration on a Titrino 784 (Deutsche Methrom, Filderstadt, Germany) equipped with a platinum double electrode. Methanol was used as a solvent and titrated to dryness with hydranal composite 5 before each measurement. Afterward the sample amount of 200 mg of milled extrudate was rapidly transferred into the titration vessel; the vessel closed and under constant stirring titrated. 2.6. PLM and DSC. Polarized light microscopy (PLM) experiments were conducted using a DMLM optical microscope (Leica Microsystems, Wetzlar, Germany) equipped with a DF320 digital camera (Leica Microsystems, Wetzlar, Germany). Differential scanning calorimetry (DSC) experiments were performed on a DSC1 (Mettler-Toledo GmbH, Giessen, Germany) coupled with a TC100MT cooling system (Huber Kältemaschinenbau GmbH, Offenburg, Germany). Nitrogen was used as the purging gas. Indium standards were employed for temperature and heat of fusion calibration. Hermetically sealed 40 μL aluminum pans and sample weights between 5 and 10 mg were selected throughout the study. Pierced lids were used in the DSC measurements to determine the API solubility. For evaluation of the Tg of “wet” formulations (i.e., formulations with the actual water content) the first cycle of the closed pan DSC measurements was used. The samples were measured from −20 to 140 °C with a heating rate of 10 °C/ min. For evaluation of the Tg of “dry” formulations (i.e., formulations with close to 0% water content), the second cycle of the open pan DSC measurements was used. The samples were measured from −20 to 200 °C, with a heating rate of 10 °C/min and a cooling rate of 10 °C/min. 2.7. Determination of Drug Solubility in Polymers by DSC. The methods reported by Yu and co-workers11,13 were applied for the determination of the solubility of the drugs in the polymers. A brief description of the experimental procedure involves first preparing the three Copovidone/Eudragit RS respective blends by cryomilling for 1 min at 10 Hz. Next, physical mixtures containing 40, 60, and 80 wt % nifedipine in the respective blends were prepared by accurately weighing the components into a milling vial and cryomilling for 10 min at 10 Hz. Each nifedipine/Copovidone/Eudragit RS mixture was held at 100 °C for 2 min on the DSC to remove moisture, followed by a slow heating rate of 1.5 °C/min from 25 to 180 °C to measure the temperature at which the API completes dissolution into the polymer matrix. Solubility temperature obtained in this fashion is also called dissolution end temperature (Tend). Subsequently, each nifedipine/Copovidone/Eudragit RS mixture was systematically annealed for 4 h below Tend, and the lowest annealing temperature after which no residual drug crystals are detected was taken as the more accurate solubility temperature. 2.8. X-ray Powder Diffraction. X-ray powder diffraction (XRPD) measurements were performed on a X’pert Pro MPD system (PANanalytical, Almelo, Netherlands) with a step size of 0.026° 2θ using Cu Kα radiation (40 kV and 40 mA). Samples were scanned on angular range of 11−25° 2θ, characteristic for the crystalline drug. Reflex analysis was conducted using the X’Pert HighScore 2.2d program from PANanalytical. All diffraction patterns have been edited by polynomial background subtraction. 2.9. Transmission Raman Spectroscopy. Transmission Raman scattering (TRS) spectra were measured with a TRS100

spectrometer (Cobalt Light Systems, Oxford, UK) equipped with an electrically cooled CCD. The wavelength of the excitation laser was 830 nm operating at 650 mW for all measurements. Laser illumination spot size of 4 mm diameter, medium lens collection optics, and 2 s exposure time were used for data acquisition. The spectra were either obtained directly from the extrudate pellets or from powder samples pressed as tablets (13 mm diameter, 500 mg, 100 kN) as suitable using an automated sample tray. In order to remove the spectral background and make differences between the spectra more clear, second derivatives of the Raman spectra were calculated. To obtain the second derivative the Savitzky−Golay algorithm was applied, using window width of 11 points and a second order polynomial for interpolation. 2.10. Drug Release. Dissolution studies were performed using an USP apparatus 2 setup (SR8 Plus, Hanson Research, Chatsworth, USA) using brown glass vessels. The cryomilled extrudates (150 mg) were manually filled into gelatin capsules. The capsules were transferred into Japanese pharmacopeia sinkers to prevent the samples from floating. Dissolution testing was conducted in 1000 mL of buffer at pH 6.8 (0.02 M Na2HPO4, 0.05 M NaH2PO4·2H2O, 0.5% SDS) and 37 °C and at a stirring speed of 100 rpm. Samples were taken after 1, 2, 3, 4, 5, 6, 7, and 8 h and analyzed by reversed-phase HPLC (see above) with UV detector. In case of the drug release experiments with pH-change by buffer addition, dissolution was started with 900 mL buffer at pH 1.2 (0.08 M HCl, 0.5% Tween80), and after 2 h, 20 mL of a K3PO4·H2O solution (2 M) was added to adjust to pH 6.8 ± 0.1. The pH was verified using a MP 230 K pH meter (Mettler-Toledo GmbH, Giessen, Germany) equipped with an InLab 410 electrode.

3. RESULTS AND DISCUSSION As a BCS II drug with a high tendency to recrystallize, nifedipine is a widely used model compound in ASD research.11,14 It is known that nifedipine is prone to crystallization above and below the glass transition temperature because of its high molecular mobility and the effect of surface enhanced crystallization.15 Samples of nifedipine formulations in three different compositions were produced in the year 1991 and investigated after 25 years of storage under ambient conditions to demonstrate the long-term stability of such thermodynamically unstable ASDs. Analytical data obtained after 25 years of storage was compared to data measured directly after production of the samples in 1991. However, at the time of initial testing in 1991 a number of advanced characterization methods were not yet available or established as hot melt extrusion for pharmaceutical formulation was only an emerging field in the early nineties. Therefore, the available original data of the investigated samples is sparse and limited to dissolution data and API content. In order to fully characterize the samples including the physicochemical properties and compare the results to the stored samples, a new batch for each of the three formulations was manufactured and analyzed. These freshly prepared batches were used to ensure a one to one comparison of analytical results of the stored samples using previously unavailable state of the art methods. The series of stored samples is designated by the letter A and the series of the freshly prepared samples by the letter B throughout the article. (Due to the technical development taking place during the last decades, the original extruder that was used to produce the nifedipine extrudates in C


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Table 2. Tg, API Content, Water Content, Water Corrected API Content, and API Content before Storage of the Samples of the A-Series

the year 1991 was no longer available. Instead, a modern twin screw extruder was used to manufacture the new batch of nifedipine extrudates. Three different extrusion temperatures were used to ensure comparability despite the change in equipment.) The freshly prepared and stored batches were investigated with pharmaceutical standard methods regarding the chemical and physical integrity of the material. Additionally, the crystalline content of all samples was investigated using different, complementary methods including PLM, DSC, XRPD, and TRS to ensure the validity of the obtained results. An overview of the sample compositions and extrusion temperatures is given in Table 1. The phase diagram of the three different formulations is depicted in Figure 1.10a 3.1. Solubility of Nifedipine in Polymer Matrixes. Due to the high viscosity of the glassy polymers typically used in ASDs, determining the solubility of an API is an analytically challenging question. Usually this question would need to be addressed by high temperature measurements significantly above the Tg of the polymer, making such measurements impractical and raising stability challenges.10a,11,16 However, the growing interest in this type of pharmaceutical formulation led to the development of a multitude of analytical methods to determine the API solubility using conventional DSC equipment.10a,13,17 In this work, the empirical solubility estimation by Kyeremateng et al. was used, as we experienced this method to be reliable as well as accurate.10a This approach allows constructing the phase diagram of a given formulation by DSC measurements and hence extrapolating the data to storage-relevant conditions. The phase diagram consisting of the solubility curve and the Gordon−Taylor fit of the T g for the three different formulations is depicted in Figure 1. Solubility curve was constructed by fitting the experimentally determined solubility data with the equation reported by Kyeremateng et al.,10a and details are provided in the Supporting Information of this article. As solubility temperature typically approaches Tg of the polymer, dissolution kinetics of the API become much slower than the time scale of the DSC measurement because of high polymer viscosity.10a Under such conditions, overestimation of the solubility temperature occurs.10a Therefore, experimental solubility temperature for API load less than 40% (w/w) was not considered for the solubility curve construction. Depending on the position relative to the solubility and Tg curve in the phase diagram, four regions can be distinguished as depicted: (i) thermodynamically stable melt, (ii) thermodynamically stable glassy solid, (iii) kinetically stable glassy solid, and (iv) the undercooled or unstable melt, respectively. As can be noted, for a 20% (w/w) drug load and a temperature of 25 °C, all the three formulations are in the kinetically stabilized region (iii) of the phase diagram. Based on extrapolation of the solubility curve, the estimated solubility at 25 °C of nifedipine in A1 is 3.7% (w/w), and in A2 and A3 matrixes is 7.9% (w/w), further indicating the supersaturation of all the investigated formulations at the given temperature. 3.2. Molecular Mobility. For a given temperature and composition, the molecular mobility of an ASD mainly depends on Tg. The lower the storage temperature is with respect to the Tg, the slower is the molecular mobility.8,18 The measured values of Tg for the stored as well as freshly prepared samples are listed in Table 2 and Table 3, respectively. Two Tg values for each sample are listed in the tables: The open pan Tg value

sample

A1

A2

A3

Tg (mean, open pan)/°C Tg (mean, closed pan)/°C content/% (w/w API) sum of impurities/% (w/w) water content/% (w/w) water corrected content/% (w/w API) content at t0/% (w/w API)

90 55 97.3 0.2 4.5 101.9 100.0

91 54 96.7 0.3 4.2 100.9 99.9

91 54 96.1 0.2 4.3 100.4 99.7

represents the glass transition of the heated out samples, excluding the influence of water. The closed pan Tg value is a measure of the actual Tg of the samples, lowered by the water uptake. The Tg (closed pan) of the stored samples is 55 °C, approximately 15 °C lower than the Tg (closed pan) of the freshly prepared samples. This difference in Tg is expected because the water content of the stored samples is approximately 2% (w/w) higher than the water content of the freshly prepared samples. The difference between Tg (closed pan) of the stored samples and storage temperature is ∼30 °C and, as such, well below the 50 °C threshold, which is assumed to be the point of significant kinetic stabilization.12 Due to the water uptake during the storage of the samples, the supersaturation of the API in the matrix increases significantly, making the stability of the samples over the storage period even more noteworthy. Another factor impacting the molecular mobility of an API in an ASD is the extent of API−polymer interaction.19 An indicator for the amount of API−polymer interaction is the Flory−Huggins parameter, accessible via the solubility data and the Flory−Huggins equation.13,20 The calculated Flory− Huggins parameter (χ) for the investigated nifedipine/ copovidone/Eudragit RS systems is −0.6 in the case of the A1/B1 and A2/B2 formulations and −0.3 for the A3/B3 formulation, respectively. A negative χ value indicates attractive API−polymer interaction, while a positive value signifies repulsive drug−polymer interaction.21 Such attractive API− polymer interactions reduce molecular mobility of the API and thus stabilize the ASD system against API recrystallization.19a 3.3. Assay and Water Content. The API content of the samples in 1991 was determined to be 100% (w/w API). After 25 years, the water corrected content of API in the samples of the A-Series is still 100% (w/w API). The sum of impurities in the stored samples is still way below 0.5% (w/w). Hence, no significant chemical degradation of the API has taken place. Even without correction for the water content the stored nifedipine samples would still be in the interval for approval of 95−105% (w/w API). 3.4. Drug Release. An important metric for the performance of an ASD is its dissolution behavior as it indicates the bioavailability of the API. The original dissolution procedure from the year 1991 used a pH change from pH 1.2 to 6.8 at 120 min by buffer addition. Dissolution data obtained by the buffer addition method directly after manufacturing of the original samples in the year 1991 was compared to dissolution data collected for the same samples after 25 years of storage (see Figure 2). With the exception of A1, the dissolution results of the stored samples are fairly comparable to the original data from 1991. For none of the investigated samples complete dissolution of the API was observed, and the overall drug D


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Molecular Pharmaceutics Table 3. Tg, API Content, Water Content, and Water Corrected API Content of the Samples of the B-Series

a

sample

B1.1

B1.2

B1.3

B2.1

B2.2

B2.3

B3.1

B3.2

B3.3

Tg (mean, open pan)/°C Tg (mean, closed pan)/°C content/% (w/w API) sum of impurities/% (w/w) water content/% (w/w) water corrected content/% (w/w API)

90 65 95.4 0.1 2.2 97.5

90 68 97.4 0.1 2.7 100.1

91 71 98.6 0.1 2.3 100.9

91 72 98.1 0.1 2.1 100.2

89 70 98.0 0.1 2.7 100.7

90 73 98.0 0.1 2.1 100.1

90 55a 96.0 0.1 2.2 98.2

91 70 98.3 0.1 2.2 100.5

90 70 98.3 0.1 2.1 100.4

The second heating cycle was used to determine the Tg of sample B3.1, as the signal was superimposed by a moisture event in the first heating cycle.

Figure 2. Dissolution data obtained by buffer addition method of the stored samples obtained at t0 and after 25 years of storage: (a) 10% (w/w), (b) 15% (w/w), and (c) 20% (w/w) Eudragit RS. The time of buffer addition is indicated by a vertical line.

Figure 3. Dissolution data at constant pH of 6.8 of all investigated samples ordered by sample composition: (a) 10% (w/w), (b) 15% (w/w), and (c) 20% (w/w) Eudragit RS.

copovidone may have led to a surface diffusion effect. This change in concentration profile of the components might have caused the observed dissolution profile. To circumvent the pH-dependent effects on the drug release of the formulations, the drug release of the stored extrudates and freshly prepared samples, was studied with a dissolution experiment at constant pH of 6.8 (see Figure 3). The overall dissolution behavior in this experiment still is dependent on the content of Eudragit RS as discussed above.22,23 A propensity to lower drug release of the API with increasing content of Eudragit RS was observed. Comparing the extrudates of the distinct extrusion temperatures does not show a clear trend in dissolution behavior. On one hand, the extrudates manufactured 25 years ago are in general at the lower end of the measured dissolution data. On the other hand, the recently manufactured extrudates do not show any systematic trend to lower or higher drug release regarding their extrusion

release decreases with increasing content of Eudragit RS in the formulation. This effect is known in the literature and originates in the physical properties of the retarding agent Eudragit RS.22 Another observation that can be linked to Eudragit RS is the influence of the pH-change on the dissolution behavior. Most evident in the t0 data of sample A1, which undergoes a rapid boost in API release after buffer addition, no meaningful impact of the pH-change is observed in all other dissolution experiments. Peculiarly, not even in the currently measured dissolution profile of sample A1 this change is observed. The cause for this observation likely is the complex, pH-dependent behavior of Eudragit RS, the interplay between dissolution, and the swelling of the polymer.22,23 A significant increase in drug release upon pH-change is not observed in the samples with higher Eudragit RS content. Taking into account the higher water content of the samples after storage, it can be speculated that the better dissolution of the well water-soluble polymer E


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Figure 4. Photographs of nifedipine extrudates produced in 1991 after 25 years of storage.

Figure 5. PLM images of samples A1−A3 and the B-samples produced at the lowest temperature. Only in the images of B1.1 and B3.1 a few birefringent structures (encircled) can be made out.

temperature: For the latter observations, the overall variability in the samples seems to be the reason since the standard deviation of the measurement is exceeding 10% (w/w API) in some cases. For the apparently lower drug release of the extrudates from 1991, the quality of the raw materials might as well be a reason to seek since processing and quality control of polymers for extrusion has gained a lot of attention during the last decades due to a rising customer demand. 3.5. Crystallinity Testing. One of the key characteristics and stability indicators of an ASD is the absence of crystallinity. Since detection and quantification of low amounts of crystallinity in pharmaceutical formulations still remains to be a challenging task, multiple complementary techniques were applied: PLM imaging was used as the extremely sensitive standard qualitative technique, XRPD was employed as the standard quantitative tool, and additionally transmission Raman spectroscopy was used as a new analytical tool probing complementary sample properties.

Already the optical appearance of the extrudates of nifedipine produced in the year 1991 is a first indicator for the absence of crystallinity: Presence of crystals or phase separation typically results in extrudate pellets to become cloudy and opaque as the extrudates are no longer completely monophasic. The pellets stored for 25 years on the contrary are still transparent (see Figure 4) indicating the absence of crystallinity and of phase separation. Further insight regarding the optical appearance of the samples was gained by PLM imaging. The PLM images of the samples A1−A3 as well as the extrudates of the B-series produced at the lowest temperature are shown in Figure 5. In the samples of the stored extrudates, no birefringent spots were detected. This indicates that not even trace amounts of crystalline API were present in these samples. The freshly prepared extrudates only showed a few spots of birefringence in the samples B1.1 and B3.1. No birefringent spots were detected by PLM in the other investigated samples (PLM images of all F


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Figure 6. Hot stage PLM images of the samples B1.1 and B3.1 at room temperature and above the melting point of nifedipine.

samples can be found in the Supporting Information). To further elucidate the nature of the birefringent spots observed in the PLM images of the samples B1.1 and B3.1, a hot-stage PLM investigation was conducted. Figure 6 shows the images obtained at ambient temperature and 180 °C, which is well above the melting point of the highest melting polymorph of nifedipine (173 °C). As the birefringent spots are still observed in the PLM images taken above the melting point of the API, it can be excluded that the birefringent spots are related to residual API crystallinity. Hence, the source of the birefringence is an inorganic impurity or cellulose introduced during the process. As only a hand full of birefringent spots is observed in the PLM images, the amount of those impurities is estimated to be well below 0.05% (w/w). Unavoidable dust and abrasion is therefore identified as the root cause of those impurities. The absence of crystalline API was further substantiated by XRPD measurements (Figures 7−9) as this analytical technique interrogates crystalline content directly. The extrudates of the A-series showed no distinct reflexes in the XRPD, which confirmed that these samples are crystalline free by this method (Figure 7). For the freshly prepared samples produced at low extrusion temperatures, some small reflexes barely above noise

were observed in the XRPD diffractrograms (Figure 8). Those patterns do not resemble the intensity profile of nifedipine and

Figure 8. XRPD patterns of nifedipine (black), B1.1 (green), B2.1 (blue), and B3.1 (red). Some distinct signals are observed in the case of the extrudates, which do not resemble the intensity pattern of the API.

rather seem to stem from impurities than from residual crystallinity of the API. This finding is consistent with the observations made by PLM discussed above. The B-series extrudates manufactured at higher temperatures showed no distinct reflexes above noise in the XRPD (Figure 9). The conducted XRPD experiments confirm that the stored extrudates of nifedipine are still crystal free after 25 years of storage. As an additional supplementary technique to detect crystallinity, transmission Raman spectroscopy was employed. As a vibrational spectroscopy method, the analytical information gathered by TRS is complementary to the optical information gathered by PLM and the X-ray diffraction pattern obtained by XRPD. TRS spectroscopy is able to capture the phonon region of the Raman spectrum below 250 cm−1, where distinct crystal signals can be detected.24 Comparing the TRS spectra of nifedipine and the A1 sample (Figure 10a), a peak at 169 cm−1 is present, which is unique for crystalline nifedipine and does not occur in the spectra of the amorphous extrudates

Figure 7. XRPD patterns of nifedipine (black), A1 (green), A2 (blue), and A3 (red). No distinct signals are observed in the case of the extrudates. G


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4. CONCLUSION In this study, kinetically stabilized amorphous solid dispersions of nifedipine were characterized regarding their chemical and physical stability as well as their dissolution performance after a quarter century of uncontrolled storage. An in-depth characterization with state of the art methods was conducted, comparing the results with a remanufactured sample batch. Still available, original dissolution data was compared to dissolution profiles collected after storage. Using the established techniques PLM and XRPD, no recrystallization could be detected in the samples after 25 years of storage. This finding was supplemented by TRS spectroscopy as a complementary analytical technique. Regarding the dissolution performance of the stored samples, only minor differences compared to the freshly manufactured extrudates could be observed. The findings presented here prove the stability of thermodynamically unstable extrudates of amorphous nifedipine even after a quarter century of storage. Although only kinetically stabilized, in the investigated case the recrystallization is inhibited over this long storage time. Even at storage temperatures of only 30 °C below the Tg, the molecular motion in the investigated ASDs is not fast enough to induce crystallization on the observed time scale. This observation shows that long-term stability much beyond the typically desired shelf life for amorphous solid dispersions is indeed possible by kinetic stabilization only, and implicating the design of HME formulations with glass transition temperatures fairly close to the desired storage conditions is indeed possible; such formulation concepts are classically considered higher risk systems. Under the observed kinetic conditions, it remains to

Figure 9. XRPD patterns of B1.2 (black), B1.3 (red), B2.2 (blue), B2.3 (magenta), B3.2 (green), and B3.3 (brown). Some signal barely above noise level is observed in all extrudates.

(TRS spectra of all other extrudates can be found in the Supporting Information). The absence of this peak therefore concludes the absence of crystallinity in the extrudates. Second derivatives of the spectra were calculated in order to remove the spectral background and highlight the differences between crystalline nifedipine and the extrudates in this particular spectral region more clearly. A comparison of the derivative spectra is shown in Figure 10b−d. None of the investigated extrudates shows a signal above baseline at 169 cm−1. Supplementing the results of PLM and XRPD, TRS spectroscopy also shows that no crystalline material is present in the investigated samples.

Figure 10. (a) TRS spectra of pure nifedipine (red) and sample A1 (blue); (b) 2nd derivative of the TRS spectra of nifedipine (black), A1 (red), B1.1 (blue), B1.2 (magenta), and B1.3 (olive); (c) 2nd derivative of the TRS spectra of nifedipine (black), A2 (red), B2.1 (blue), B2.2 (magenta), and B2.3 (olive); and (d) 2nd derivative of the TRS spectra of nifedipine (black), A3 (red), B3.1 (blue), B3.2 (magenta), and B3.3 (olive). In the phonon region, no signal of crystalline API can be detected for all investigated extrudates. H


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be speculated whether even residual crystallinity would have an impact on crystal growth in such ASDs.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00783. PLM images, TRS spectra, and solubility curve fitting (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 621 589 2526. Fax: +49 621 589 3092. E-mail: gerd. [email protected]. ORCID

Frank Theil: 0000-0002-9396-3118 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS All authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

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REFERENCES

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