Molecular Interactions in Posaconazole Amorphous Solid Dispersions

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Molecular Interactions in Posaconazole Amorphous Solid Dispersions from Two-Dimensional Solid-State NMR Spectroscopy Xingyu Lu, Chengbin Huang, Michael B. Lowinger, Fengyuan Yang, Wei Xu, Chad D Brown, David Hesk, Athanas Koynov, Luke Schenck, and Yongchao Su Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00174 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Molecular Pharmaceutics

Molecular Interactions in Posaconazole Amorphous Solid Dispersions from Two-Dimensional Solid-State NMR Spectroscopy Xingyu Lu1, Chengbin Huang1,2, Michael B. Lowinger1, Fengyuan Yang1,3, Wei Xu1, Chad D. Brown1, David Hesk1, Athanas Koynov1, Luke Schenck1, Yongchao Su1,* 1

Merck Research Laboratories (MRLs), Merck & Co., Inc., Kenilworth, New Jersey, 07033, USA 2

School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin, 53705, USA 3

Ashland Inc., Wilmington, Delaware 19808, USA

Revised for Molecular Pharmaceutics April 03, 2019

Running title: Molecular Interaction in Posaconazole Amorphous Solid Dispersions * Corresponding author: Y.S.: [email protected]

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

Table of Content (TOC) Figure


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Abstract Molecular interactions between the active pharmaceutical ingredient (API) and polymer have potentially substantial impacts on the physical stability of amorphous solid dispersions (ASDs) presumably by manipulating molecular mobility and miscibility. However, structural details for understanding the nature of the molecular contacts and mechanistic roles in various physicochemical and thermodynamic events often remain unclear. This study provides a spectroscopic characterization of posaconazole (POSA) formulations, a second-generation triazole antifungal drug (Noxafil®, Merck & Co., Inc., Kenilworth, NJ, USA), at molecular resolution. One- and two-dimensional (1D and 2D) solid-state NMR (ssNMR) techniques including spectral editing, heteronuclear 1H-13C, 19F-13C, 15N-13C and 19F-1H polarization transfer and spin correlation and ultrafast magic angle spinning (MAS), together with the isotopic labeling strategy, were utilized to uncover molecular details in POSA ASDs in a site-specific manner. Active groups in triazole and difluorophenyl rings exhibited rich but distinct categories of interactions with two polymers, hypromellose acetate succinate (HPMCAS) and hypromellose phthalate (HPMCP), including intermolecular O-H···O=C and O-H···F-C hydrogen bonding, π-π aromatic packing and electrostatic interaction. Interestingly, the chlorine-to-fluorine substituent in POSA, one of the major structural differences from itraconazole that could facilitate binding to the biological target, offers an additional contact with the polymer. These findings exhibit 2D ssNMR as a sensitive technique for probing subnanometer structures of pharmaceutical materials and provide a structural basis for optimizing the type and strength of drug-polymer interactions in the design of amorphous formulations. Keywords Amorphous Solid Dispersions; Fluorinated Pharmaceuticals; Posaconazole; Molecular Interaction; Solidstate NMR; Two-dimensional Spectroscopy



1. Introduction Amorphous solids have been widely utilized in the pharmaceutical development of poorly soluble drugs for the enhanced aqueous solubility over the crystalline counterpart. To prevent drug recrystallization over time scales of pharmaceutical interest, active pharmaceutical ingredients (APIs) are usually formulated with polymer additives as amorphous solid dispersions (ASDs) to enhance physical stability against crystallization. For example, an addition of only 1 wt % polyvinylpyrrolidone (PVP) can slow down the crystal growth rate of amorphous nifedipine by 10 times.1 Molecular properties, e.g., polymer segmental mobility relative to drug dynamics1-4 and various API-API, API-polymer as well as API-excipient interactions5-12, are found to be critical to physical stability of amorphous drugs. Among these factors, previous studies have suggested a general correlation between API-polymer interactions and the inhibition effect of polymer on the crystallization of drugs.6, 7, 11, 13-15 This correlation, very importantly, provides a prediction for optimizing polymeric excipients to enhance the stability of amorphous formulations. Therefore, structural details in ASDs are critical for uncovering mechanistic roles in the interplay of molecular contacts, molecular mobility and physical stability. Understanding the API-polymer interaction requires comprehensive characterizations of ASDs at a molecular level. Many techniques have been utilized to characterize ASDs including scanning electron microscopy (SEM), differential scanning calorimetry (DSC), infrared spectroscopy (IR), Raman spectroscopy and powder X-ray diffraction (PXRD)16. As a pioneer effort, Taylor and Zografi have utilized IR and Raman to characterize interactions in indomethacin-polyvinylpyrrolidone (PVP) and lyophilized sugar-PVP amorphous dispersions in the 1990s.17,

18

Since then, many studies have employed these

techniques to investigate API-polymer interactions. For example, Marsac et al. studied the interaction between felodipine and PVP by using Fourier-transform-IR analysis and correlated it with the drug crystallization kinetics at various temperatures.5 Meng et al. investigated the interaction between curcumin and four hydrophilic polymers using FT-IR and Raman.19 Kothari et al. carried out an FT-IR study on the interaction between nifedipine and various polymers11. Their results correlated the strongest API-polymer hydrogen bonding interaction with the highest resistance to drug crystallization. Recently, De Araujo et al. utilized high-energy X-ray diffraction at Advanced Photon Source (APS) and pair distribution function analysis to isolate the API-API and API-polymer interactions in ASDs 20, while the chemical information is still lacking. These studies highlighted the critical role of molecular interaction on the physical stability. However, it is still technically challenging to determine the atomic level characteristics of ASDs due to the lack of long-range order in amorphous materials. Knowledge of the pattern and strength of intermolecular contact between API and polymer, i.e., the chemical nature of the interaction type and involvement of


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functional groups, is still limited. Therefore, there is a strong need for high-resolution characterizations of ASDs from a molecular perspective. Solid-state nuclear magnetic resonance (ssNMR) is a high-resolution analytical technique which provides atomic information on molecular structure, dynamics, and domain morphology in small molecule and macromolecular systems.21-24 Most previous ssNMR applications to pharmaceutical sciences analyzed interactions in ASDs by observing changes to chemical shift and relaxation time. These NMR parameters reflect local structural changes, e.g., chemical shielding or deshielding and molecular motions, and can be measured using mainly one-dimensional (1D) or pseudo two-dimensional (2D) magic angle spinning (MAS) techniques. For example, Munson and coworkers successfully identified and quantified API-API and APIpolymer interactions in amorphous indomethacin and ASDs dispersions using the 1D 13C cross polarization (CP) method.9 13C isotopically labeling at the carboxylic acid carbon of indomethacin significantly helped to selectively study the peaks of interest. Lubach and coworkers characterized lapatinib-HPMCAS interactions in ASDs from changes of 1D 15N chemical shifts.7 Moreover, the 1D 15N spectral comparison has provided spectroscopic evidence for protonation of lapatinib (i.e., salt formation) in the polymer matrix. Suryanarayanan and coworkers investigated the effects of ionic and hydrogen bonding interactions on the molecular mobility of ketoconazole in ASDs by utilizing 1D

C and

13

N CP-MAS NMR and density

15

functional theory (DFT) calculation.6 Their results further suggested that stronger API-polymer interactions can potentially delay the crystallization onset temperature as well as crystallization tendency by reducing the molecular mobility. These studies showcased 1D 13C and 15N ssNMR as a powerful tool for detecting molecular interaction in amorphous dispersions. However, many scenarios may either go beyond the capability of the 1D ssNMR method or need a more sensitive and direct measurement. Firstly, relatively weak interactions may not induce observable spectral changes. Secondly, one needs to consider many other factors including molecular packing, conformational change, and water binding, that could perturb peak positions, in addition to molecular interactions. Thirdly, 1D spectra often exhibit limited resolution due to the peak overlaps. These concerns may apply to IR and Raman measurements as well. For ssNMR, disruptive regimes of molecular dynamics, magnetic field limitation and inefficient homonuclear couplings can often diminish the opportunity to observe resonance changes of the peak of interest in crowded 1D spectra. As a result, these spectroscopic limitations result in challenges with unambiguous peak assignments, hindering a robust analysis. Consequently, an advanced spectroscopic method is needed to provide a more direct, sensitive and site-specific characterization of critical interactions in ASDs. By extending 1D ssNMR to an additional homonuclear or heteronuclear dimension, 2D spectroscopy provides a better resolution for resolving peak overlapping issue. Most importantly, cross



peaks in 2D spectra, e.g. of 1H-13C heteronuclear correlation (HETCOR), are a spectroscopic consequence of internuclear spatial proximity. Therefore, 2D ssNMR methods serve as a high-resolution and reliable approach to probe interactions between drug substances and polymers in amorphous dispersions, as seen in few previous studies.8, 25-28 For example, Vogt and coworkers utilized 2D 13C-1H and 19F-1H correlation experiments to characterize associations between the amorphous drugs of acetaminophen as well as indomethacin and different polymer additives. The study also investigated interactions between adsorbed ezetimibe and silica through

29

Si-1H and

Si-19F heteronuclear correlation28. New methodological and

29

technical advancements in recent years offer novel applications of these measurements as well as additional structural information. For example, reliable resonance assignments are critical for extracting site-specific information in understanding the chemical nature of molecular interactions. However, previous 2D ssNMR investigations of amorphous pharmaceutical materials often lack unambiguous peak identification. Advanced techniques including ultrafast MAS, dynamic nuclear polarization (DNP), and multidimensional spectroscopy have emerged and exhibited great potential in their application to structural biology, material and pharmaceutical sciences.21, 23, 24, 29-32 Recent studies have successfully utilized the enhanced sensitivity and resolution from proton-detected spectroscopy at 60-110 kHz MAS13, 32 and DNP-enhanced ssNMR2931, 33

for analyzing amorphous formulations, and also demonstrated the feasibility of full spectral

assignments for amorphous APIs by utilizing spectral editing and multi-dimensional, multi-nuclear and multi-quantum correlations34,

35

. Moreover, reference spectra of amorphous APIs and polymers are

necessary to unambiguously identify intermolecular API-polymer interactions in ASD spectra, which may be challenging given the practical lack of availability of physically stable amorphous API samples. In the present study, we utilized posaconazole (POSA) as a model to investigate the molecular interaction of a fluorinated drug molecule with pharmaceutical polymers in amorphous solid dispersions. POSA is a second-generation triazole antifungal drug (Noxafil®, Merck & Co., Inc., Kenilworth, NJ, USA) and functions by disrupting the bilayer packing of phospholipids and interacting with membrane-bound complexes.36, 37 Crystalline POSA has a low solubility of < 0.1 µg/ml at pH > 4. To overcome the challenge of its poor aqueous solubility, the commercial product was formulated as an amorphous solid dispersion to enhance oral bioavailability. Samples of amorphous POSA and its ASDs with two cellulose-based polymers, HPMCAS and HPMCP, were prepared by using melt-quenching or spray drying techniques. We used 2D ssNMR spectroscopy to provide site-specific characterizations of API-polymer interactions. We successfully demonstrated direct observation of API-polymer intermolecular correlations to differentiate the types of molecular interactions between the drug and the two polymers utilized in this study. 2. Samples and Methods


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Materials Crystalline POSA was provided by Merck & Co., Inc., Kenilworth, NJ, USA. The schematic molecular structure of POSA is shown in Figure 1A. Isotopically 13C and 15N labeled POSA is synthesized for site-specific investigations by ssNMR. The 13C and 15N enriched sites are shown in red in Figure 1B. HPMCAS (LF) was obtained from by Ashland Inc. USA. HPMCP HP50 was purchased from Shin-Etsu Chemical Co., Ltd. Japan. Organic solvents including methanol and acetone were purchased from Sigma Aldrich (St. Louis, MO). All compounds were utilized as-received for preparing ASDs. Amorphous solid dispersions prepared by melt-quenching and spray-drying Physical blends of POSA and HPMCAS at different drug loadings from 10 to 90 wt % were prepared. Each sample was cryo-milled (SPEX CertiPrep 6750 with liquid nitrogen as coolant, Metuchen, NJ) at 10 Hz for five 2 minutes cycles, each followed by a 2-minute cooldown. Pure API and polymer samples were processed using the same technique. Melt-quenched POSA-HPMCAS ASDs were prepared by melting the cryo-milled drug-polymer mixtures at 453 K for 20 min (thermally stable based on the previous study38), followed by grinding using a mortar and pestle for subsequent ssNMR measurements. The 13C/15N isotopically labeled POSA sample at a drug concentration of 30 wt % containing HPMCAS was prepared by hand-milling for the limited quantity of labeled POSA, followed by melting and cooling. A spray-dried POSA-HPMCP ASD at 30% drug loading was prepared on a ProCepT 4M8-TriX spray dryer equipped with a 0.8 mm two-fluid nozzle (Procept, Zelzate, Belgium). Briefly, HPMCP and POSA were dissolved in a methanol/acetone (1:1 v/v) co-solvent at a total solid concentration of 20 mg/mL. The solution was then sprayed at a rate of 5.8 mL/min and atomized using compressed air at 70 psi. The drying air was maintained at a flow rate of 0.1 m3/min with inlet and outlet temperatures of 363 and 323 K, respectively. Differential scanning calorimetry DSC measurements were carried out with a TA Q2000 (TA Instruments Inc., USA) under 50 mL/min N2 purge in hermetically sealed aluminum pans. The glass transition temperature was determined from the onset temperature of the heat flow inflection point obtained following a method of heating the sample at 10 K/min, followed by cooling at 10 K/min. Raman Raman spectra of neat POSA, neat polymers and POSA-polymer dispersions were collected through a Raman microscope (Thermo Scientific DXR Raman microscope). A 10 mW 532 nm laser was



utilized for excitation. Background, fluorescence and white-light corrections were applied. Raman spectra were collected on a translational stage at 32 numbers of scan for neat POSA and 512 numbers of scan for neat polymers as well as POSA-polymer dispersions. Each exposure of 5 seconds was performed at the spectral range 100 – 3500 cm-1 at 5 cm-1 resolution. Solid-state NMR spectroscopy MAS NMR experiments were carried out using Bruker Advance III HD 400 and Advance III 500 spectrometers operating at proton frequencies of 399.87 and 500.13 MHz, respectively, in the Biopharmaceutical NMR Laboratory (BNL) at Pharmaceutical Sciences, MRL (Merck & Co., Inc., West Point, PA, USA). All experiments were conducted with a MAS frequency of 12 kHz at 298 K except spectra acquired under ultrafast MAS at 60 kHz. Most 13C and 19F ssNMR spectra were obtained with a Bruker 4 mm HFX MAS probe in triple-resonance mode tuned to 1H, 19F, and 13C frequencies or in double-resonance mode tuned to 1H and 13C frequencies. For experiments at an ultrafast MAS frequency of 60 kHz, a 1.3 mm H/19F/X probe (X tuned to

1

13

C) was utilized. Samples were packed in 4.0 mm and 1.3 mm rotors for

experiments with MAS frequencies of 12 kHz and 60 kHz, respectively. 1H and 13C spectra were referenced to tetramethylsilane (TMS). 19F spectra were referenced to Teflon at -122 ppm. For experiments at 12 kHz MAS, 1D 13C-1H and 13C-19F CP transfers were carried out at a radio-frequency (RF) strength of 80-100 kHz. The power level was ramped linearly during the contact time over a depth of 15 to 20 kHz on the 1H or 19F channel to enhance CP efficiency. 100 kHz SPINAL-64 1H and 19F heteronuclear decoupling was applied during 13C detection. Besides routine 13C CP MAS experiments, edited 13C spectra were acquired for identifying the carbon multiplicity. The 13C-15N double-CP transfer was performed on the isotopically labeled sample. It started with a 1H to 15N CP transfer then followed by a 15N to 13C CP transfer to detect the carbons neighbored to 15N. All data were processed in Bruker Topspin 3.5 software, Gaussian and Qsine-bell lineshape broadenings were applied to achieve sensitivity and resolution enhanced spectra for structure analysis. 2D heteronuclear dipolar correlation spectra between 1H and 13C (or 19F) nuclei were acquired using CP contact time ranging from 100 μs to 2 ms. When the contact time is relatively short, e.g., on the order of 100 μs, it can selectively establish short-range 1H-13C dipolar correlations. In contrast, a long 2-ms CP contact time presumably provides 1H-13C connectivity up to 5 Å. The spectral features of API and polymer make ASDs suitable molecular systems to utilize the 2D HETCOR method. For example, most APIs have aromatic groups which exhibit 1H chemical shift ranging from around 5 to 10 ppm and 13C chemical shift ranging from around 100 to 160 ppm. Functional groups of pharmaceutical polymers, e.g., HPMCAS, often reside in aliphatic regions where the 1H chemical shift ranges from 0 to ~ 5 ppm and 13C chemical shift


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ranges from 0 to ~ 100 ppm. Therefore, the distinct chemical shift distribution between API and polymer provide spectral difference for identifying intermolecular API-polymer contacts in a 2D manner. The relatively long intermolecular 1H–13C transfer can be established using both 13C-detected (the 4 mm probe and 12 kHz MAS) and 1H-detected (the 1.3 mm probe and 60 kHz MAS) HETCOR experiments. 3. Results

Figure 1. Schematic structures of POSA (A), the local chemical groups of 13C, 15N - isotopically labeled POSA (B), HPMCAS (C) and HPMCP (D). Labeled atoms of C44, N42, and N43 are highlighted in red. We initially carried out preliminary solid-state characterization on POSA, polymers, and ASDs using DSC, Raman spectroscopy and 1D ssNMR, and then proceeded with in-depth investigations on the nature of various API-polymer interactions using 2D ssNMR techniques. Near-complete assignments of carbons and protons of amorphous POSA and polymers are achieved by utilizing 1D and 2D spectral-edited experiments described previously.34 Magnetization transfers of both 13C-1H and 19F-13C pairs were utilized for establishing the heteronuclear correlations. All POSA-HPMCAS ASD samples were prepared via meltquenching for investigating the structural details of amorphous formulations produced at elevated temperatures. The thermal stability of HPMCP could not allow a successful production of POSA ASD at the desired temperature. Therefore, one 30 wt% drug-loaded POSA-HPMCP ASD was prepared via spray drying for a comparison study. These samples and experiments were designed for understanding the nature



of molecular interactions and for evaluating the sensitivity and capability of heteronuclear 1D and 2D ssNMR techniques on examining the local molecular environment of amorphous formulations. 3.1. Characterizations of Amorphous POSA and ASDs

Figure 2. (A) 1D 13C CP-MAS spectra of crystalline and amorphous POSA, HPMCP and HPMCAS (LF). (B) Top: DSC traces of the glass transition event in POSA-HPMCAS dispersions; and bottom: the glass transition temperature, Tg, against the drug loading of POSA. (C) Comparison of Raman spectra among (top) melt-quenching POSA, HPMCAS, 30 wt % drug-loading ASD and (bottom) spray-drying POSA, HPMCP, 30 wt % drug-loading ASD. Spinning sidebands of carbonyl resonances may show minor intensities in (A). The molecular structure of POSA is shown in Figure 1A, with carbon and nitrogen atom numbers labeled. As a weak base compound, POSA has a rich chemical structure including hydroxyl and carbonyl groups and triazole as well as difluorophenyl rings. HPMCAS and HPMCP contains 4-5 different types of sidechain groups including the carbonyl and aromatic ring, as shown in Figure 1C and D. These sidechain groups exhibit potential to have distinct intermolecular contacts with POSA. 1D 13C CP-MAS spectra of crystalline and amorphous POSA, HPMCP and HPMCAS, are shown in Figure 2A. The broad peaks of melt-quenched POSA confirm its amorphous nature, which agree well with the powder x-ray diffraction results (data not shown). The preliminary carbon assignments for HPMCAS and HPMCP show characteristic carbonyl peaks around 170 ppm, aromatic peaks at 130 ppm (for HPMCP), and various CH2 and CH peaks from around 50 ppm to 110 ppm. The only carbonyl peak of POSA, C41, appears at around


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153.2 ppm as identified in the spectral assignment in the next sessions and is not overlapped with C=O peaks of polymers. To evaluate the POSA-HPMCAS miscibility and interaction in melt-quenched ASDs, we respectively utilized DSC and Raman spectroscopy for preliminary characterizations. DSC analysis was applied to examine the miscibility of POSA-HPMCAS ASDs prepared at various drug loadings from 10% to 90% (w/w). Figure 2B shows DSC traces of the glass transition event in POSA/HPMCAS dispersions. Each dispersion shows a single glass transition temperature (Tg), and Tg values of POSA-HPMCAS dispersions systematically decrease with increasing POSA drug loading. The Tg values of POSA-HPMCAS dispersions are plotted against POSA concentration in Figure 2B, and a correlation is observed. Each dispersion exhibits a single glass transition temperature (Tg) and Tg values of dispersions systematically decrease with increasing POSA concentration. The DSC results suggest that POSA and HPMCAS are miscible in a wide range of drug concentrations. It is also worth to note that the Couchman-Karasz Equation can well describe the Tg data (Figure S1)39. In addition, Raman spectroscopy has been utilized to characterize molecular information of pharmaceutical materials.17, 19 In this study, we employed it to examine amorphous POSA and ASDs as shown in Figure 2C. The Raman spectrum of the POSAHPMCAS ASD (melt-quenched at 30 wt % drug loading) exhibits peaks representing a combination of the melt-quenched amorphous POSA and HPMCAS spectra. Similarly, the POSA-HPMCP ASD (spray-dried at a 30 wt % drug loading) spectrum exhibits peaks representing a combination of the spray-dried POSA and HPMCP spectra. Polymer peak assignment is ambiguous, such the 850-1500 cm-1 range usually representing both C-O and C-C stretching.40 Raman spectrometry is often utilized to detect molecular interaction by observing wavenumber shifts. However, there was no apparent shift (> 5 cm-1) for POSA and polymer components in all ASDs examined in this study. To continue investigating the molecular interaction, we carried out a series of 1D ssNMR experiments. Chemical shift assignments are critical for unambiguous analysis of ssNMR results. Therefore, we first accomplished the full carbon assignment by utilizing a protocol described previously.34 Briefly, 1D spectral edited experiments utilize various internuclear 1H-13C, 19F-13C, 15N-13C and 19F-1H transfers for identifying atomic connectivity. 1D 13C CP spectra can extract rich structural information by employing different pulse sequences. For example, the regular 13C CP experiment with long contact time, as shown in Figure 3A, provides a full spectral view of all carbons in the structure, including approximately 10 aliphatic carbons (chemical shift ranging from 0-100 ppm) and 20 aromatic and quaternary carbons (chemical shift ranging from 100-180 ppm). To tackle the peak overlapping issue, we utilized the 13C-edited experiments which selectively excite signals of desired carbons by manipulating the spin evolution of different



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multiplicities of CHn (n = 0-3) groups.41 One example of the 13C-edited spectra is shown in Figure 3B where the CH signals have vanished, the CH2 peaks are negative, and the quaternary carbons appear positive. Another 13C-edited spectrum in Figure 3C exhibits vanished CH2 and quaternary carbon intensity, but only shows the CH peaks. The combination of these CHn multidisciplinary spectra differentiates carbon resonances for assignment purpose.

Figure 3. 1D CP-MAS and 2D HETCOR experiments for resonance assignments of amorphous POSA. (A) The 1H-13C CP spectrum of amorphous POSA acquired with long contact time of 2 ms; (B) 1H-13C CP spectrum edited for -CH2 peaks (red) and -C peaks (black); (C) 13C-1H CP spectrum edited for -CH peaks (blue); (D) 1D 13C-19F CP spectrum with a short 100 µs contact time for extracting the intramolecular 19FC contacts; (E) 1D 1H-15N-13C double CP experiment of POSA for assigning the nearby C44 and C46

13

resonances. Note that N42 and N43 are

N labeled and C44 is

15

C labeled; (F) 2D

13

C-1H HETCOR

13

spectrum with a 2 ms contact time; (G) 2D 19F-1H HETCOR spectrum. C-F and N-H groups in the POSA molecule are potentially involved in the intermolecular interaction with polymers and thus are of great interest in this study. Carbons in 19F- or 15N-containing heterocyclic rings exist in a peak-crowded spectral area at around 100 ppm to 150 ppm and are ambiguously assigned in 1D spectral-edited 13C spectra. To extract site-specific information of these heterocyclic rings, we have applied 19F-edited and 15N-edited 13C CP-MAS experiments. In Figure 3D, the 19F-13C CP shows


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carbon atoms near the two fluorine atoms, including the adjacent 19F-bonded carbons (C15 and C17), the 2- to 4-bond distant carbons (C16, C14), and even a long-distance transfer from fluorine to C5. To assign 13C peaks in the triazole ring of POSA, we utilized the 13C and 15N isotopically labeled POSA, which was 13C-labeled at C44, and 15N-labeled at N42 and N43 (Figure 1D). The 1D double CP spectrum (Figure 3E) has three 13C resonances, the magnetization of which is transferred from 15N-labeled N42 and N43. It includes a strong peak at around 135.3 ppm, which was assigned to be 13C-labeled C44. Interestingly, we also observed natural abundance C41 (~153 ppm) and C46 (~62 ppm), the neighbored carbons to the 15N-labeled N42. Therefore, the isotopic labeling and 15N-13C double CP transfer experiment allowed us to successfully assign C41 in the amide ring and C46 in the aliphatic region, which otherwise would be technically challenging due to peak overlapping. Moreover, the carbon assignments obtained from various 1D spectra were observed in the 2D 13C-1H correlation spectrum and correlated well with proton chemical shifts, as shown in Figure 3F (right). The 19F-1H 2D HETCOR spectrum in Figure 3F (left) showed a strong correlation with a 1H chemical shift at 7.2 ppm, a contact between the two fluorine atoms and nearby protons attached to C16, C18, and C19. 3.2. POSA-HPMCAS interaction from 1D and 2D MAS NMR

Figure 4. (A) 1D 1H-13C CP MAS spectra of POSA-HPMCAS ASDs with drug loadings of 100%, 90%, 70%, 50%, 30%, 10% and 0% (top to bottom). Enlarged spectral regions exhibiting -C=O of HPMCAS (B), C41 (C) and C44 (D) of POSA. All spectra were acquired at MAS frequency of 12 kHz with 2 ms contact time.



Theoretically, the drug loading (DL) can be varied during drug development, which is often below the equilibrium solubility of the crystalline drug in the polymer for maintaining the thermodynamic stability of amorphous dispersions. In this study, we prepared a series of POSA-HPMCAS ASDs at different DLs to investigate the impact of the drug-to-polymer ratio on molecular interactions. Resonance assignments of POSA and polymer from 1D and 2D ssNMR results allowed robust analysis of the structure and interactions in POSA-polymer ASDs. 1D 13C CP-MAS has often been employed to observe molecular interactions in pharmaceutical dispersions by detecting chemical shift changes.6, 7 In this study, we utilized 1D experiments to preliminarily examine spectroscopic behaviors of ASD samples at various drug loadings and summarized the results in Figure 4. All amorphous dispersions exhibited broad peaks for the long-range disordered structure. Most peaks in the spectra remained at the same position for different drug-loaded samples while a few resonances exhibited chemical shift changes. For example, POSA C3 (~ 39.6 ppm) and polymer CH2- (~ 29.5 ppm), as guided by the dashed lines, aligned well among all ASDs. Interestingly, the carbonyl peak of HPMCAS (~172.5 ppm for the polymer-only sample) is deshielded at higher drug loadings as shown in Figure 4B. A similar phenomenon was reported in previous studies of drug-polymer dispersions using 1D ssNMR6, 7, where electrostatic interaction was proposed.

Figure 5. 2D

13

C-1H HETCOR spectra of POSA (A), HPMCAS (B) and 30 wt% drug-loaded POSA-

HPMCAS ASD (C). All spectra were acquired at MAS frequency of 12 kHz with 2 ms contact time.


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Moreover, two carbon peaks of POSA exhibited peak shifts as respectively shown in Figure 4C and 4D. As discussed in Figure 3E, the 15N-13C double CP experiment with site-specifically labeled strategy helped to assign C41 and C44 (respectively ~153.2 ppm and ~135.3 ppm for the amorphous POSA). The peak shift for C41 and C44 carbons suggests structural perturbation, although the cause of the spectral changes remains unknown. Nevertheless, 1D results suggest interesting local interactions for a few specific chemical groups and their dependence on the polymer (or drug) weight percentages. To understand the possible interaction at a molecular level, i.e., roles of chemical groups in drug substance and polymer, we continued the study by examining space proximity among 1H, 13C and 19F atoms in advanced 1D and 2D ssNMR experiments.

Figure 6. 2D 13C-1H HETCOR spectra of isotopically 13C,15N-labeled POSA (A), 30 wt% drug-loaded C,15N labeled POSA-HPMCAS ASD (B), and HPMCAS (C). All 2D spectra were acquired at 12 kHz

13

MAS with 2 ms contact time. In contrast to the approach of 1D 13C CP experiments which identify structural changes by detecting chemical changes, 2D

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C-1H heteronuclear correlation (HETCOR) techniques directly observe

intermolecular contacts between API and polymer. Presumably, an API-polymer cross peak suggests an intermolecular distance < 5.5 Å in a 2D HETCOR spectrum acquired with a relatively long 2-ms 1H-13C CP transfer26, 28. 30 wt% drug-loaded samples were utilized for these in-depth 2D ssNMR characterizations for the high relevance to ASD formulations as well as reasonable spectral sensitivity of POSA. A series of 2D spectra of amorphous POSA, HPMCAS and the ASDs were collected and are shown in Figure 5. Compared to the API (blue) and polymer (red) reference spectra, a new 13C-1H cross peak at ~153.4 ppm / 2.2 ppm appears in the ASD spectrum in Figure 5C. The fact that this peak does not exist in the reference spectra suggests it as an intermolecular POSA-HPMCAS contact. Based on the peak assignment, this carbon peak at 153.4 ppm in the 13C dimension belongs to POSA C41, a carboxyl carbon. As observed in



the 2D 13C-1H spectrum of HPMCAS, the proton peak at 2.1 ppm could be assigned as a -CH- adjacent to a hydroxyl group. Therefore, these results suggest a hydrogen bonding interaction between the POSA triazole ring and the HPMCAS hydroxyl group. The formation of H-bond moves the chemical shift of the carboxyl carbon to be more shielded, which agrees well with the observation of the POSA C41 peak shift in the 1D titration analysis in Figure 4C. Besides the hypothetical H-bond interaction at the specific site of POSA C41, another interesting intermolecular contact is detected by utilizing the 13C and 15N-labeled POSA. Figure 6 shows the 2D 13CH HETCOR spectra of amorphous POSA (13C-labeled at C44), HPMCAS and the ASD (prepared using

1

isotopically labeled POSA). A strong intramolecular C44-H44 cross peak of POSA was observed in Figure 6A for the enriched 13C abundance. The new intermolecular cross peak at ~136.2 ppm / 3.6 ppm, in addition to the C44-H44 cross peak, was observed in the ASD spectrum (Figure 6B). The 1H chemical shift of this cross peak suggest an aliphatic proton of HPMCAS, which is preliminarily assigned to a CH2 group adjacent to the carboxyl groups (Figure 6C).

Figure 7. Hypothetical structural models of POSA-HPMCAS interaction in the melt-quenching ASD: (A) hydrogen bonding; and (B) electrostatic interaction. Two interesting POSA-HPMCAS intermolecular contacts were observed in Figure 5 and 6, suggesting distinct nature of the interactions as illustrated in Figure 7. Briefly, the involvement of different chemical groups demonstrates two kinds of contacts including a hydrogen bonding interaction between the POSA triazole ring and the HPMCAS hydroxyl group (Figure 7A), and an electrostatic interaction between the POSA carbonyl group and the HPMCAS carboxyl group (Figure 7B). The H-bond of O-H···O=C between drug substance and polymer has been previously observed in ASDs.11 Moreover, in a study by Suryanarayanan and coworkers, the API-polymer electrostatic interaction between ketoconazole and poly(acrylic acid) (PAA) was proposed based on 1D 13C and 15N ssNMR and DFT calculations.6


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Figure 8. Intermolecular interaction in POSA-HPMCAS ASD observed from 1D CP-MAS spectra. (A) C-1H CP of amorphous POSA; (B) 13C-1H CP of HPMCAS; A 2 ms long contact time has been utilized

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for (A) and (B). (C) 19F-13C CP of 30 wt% drug-loaded POSA-HPMCAS ASD; Top: short CP and bottom: long CP. (D) A proposed fluorine-containing hydrogen bonding model. In addition to the 1D and 2D 13C-1H transfer and correlation techniques, 19F-13C CP experiments have successfully identified a third kind of POSA-HPMCAS interactions. Figure 8A and B respectively include 13C-1H CP spectra of amorphous POSA and HPMCAS as references. The 19F-13C CP spectrum of the 30 wt% drug-loaded POSA-HPMCAS ASD is shown in Figure 8C, where the intensity of carbon peaks was transferred from the two fluorine atoms of POSA. Compared to the POSA reference spectrum in Figure 8A, several carbon peaks including POSA C5, C14, C15, C16, C17, C18 and C19 appear in both short and long 19F-13C CP spectra, as a result of the intramolecular transfer. One interesting peak at ~ 61.2 ppm is not present in the short 19F-13C spectrum but shows up in the long CP spectrum. This peak does not originate from POSA carbons but matches well with a peak of HPMCAS as guided by the thick dashed line. This polymer peak can be tentatively assigned as the carbon neighbored to a hydroxyl group in the polymer. At the given 19F-13C CP contact time of 2 ms, an intermolecular distance of < 5.5 Å can be reasonably assumed for any observable transfer. This also agrees with the observation that only those carbons close to 19F atoms are seen, via intramolecular 19F-13C transfer. Therefore, a model of hydrogen bonding between the POSA fluorine atom and a polymer hydroxyl group is proposed in Figure 8D based on their proximity. Previous studies have shown O-H···F-C hydrogen bonds in various cases of small organic molecules42-48, and wide utilization of fluorine substituents for lead optimization in drug discovery.49



3.3. POSA-HPMCP interaction from two-dimensional MAS NMR Pharmaceutical polymers are utilized in formulation design for various stabilizing, coating and controlled-releasing applications. The investigation of interactions between drug substances and different polymers provides information about these critical molecular contacts to facilitate the understanding of pharmaceutical properties including physical stability and dissolution profiles50-52. For this reason, we extended the molecular investigation to ASD samples prepared using a different polymer. It is of particular interest to study HPMCP, which owns different substituents to HPMCAS and might interact differently with POSA. HPMCP is often used as an enteric coating agent to prevent drug degradation in gastric acid or protect the gastric mucosa from irritating drugs. Herein, we prepared a POSA-HPMCP ASD via spray drying and at the same drug loading of 30 wt% as the POSA-HPMCAS ASD samples in 2D ssNMR investigations.

Figure 9. Molecular interaction between POSA and HPMCP observed in 2D heteronuclear correlations. 2D

C-1H HETCOR spectra of amorphous POSA (A), HPMCP (B), and 30 wt% drug-loaded POSA-

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HPMCP ASD (C). (D) Two API-polymer interaction models proposed from the 2D ssNMR results.


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Figure 9 shows the 2D 13C-1H HETCOR spectra of POSA and HPMCP, and the spray dried ASD (30 wt% drug loading). The spectral comparison reveals two interesting cross peaks in the ASD spectrum at ~168 ppm/3.6 ppm and ~130 ppm/1.9 ppm (in black circles), respectively. The first cross peak is a new intermolecular cross peak between HPMCP aromatic carbons and POSA aliphatic protons. Based on the C-1H correlation in the POSA spectrum, the chemical shift at ~ 1.9 ppm matches well with protons

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attached to C46 and C47. This aliphatic chain is connected to the triazole ring. Therefore, we propose a ππ aromatic stacking model, highlighting the interaction between the HPMCAS aromatic ring and POSA the triazole ring (on the right of Figure 9D). The second interesting cross peak exhibits a shift of ~ 2.4 ppm for the carbon peak of the polymer carbonyl, indicating an interaction at the C=O site. A possible explanation is an electrostatic interaction between HPMCP carbonyl group and POSA triazole ring, as hypothesized on the left of Figure 9D. 4. Discussion 4.1. Rich structures of POSA-polymer interactions in ASDs The design of amorphous dispersions involves a balance of processability, chemical compatibility, physical stability and dissolution profile. As the major component in amorphous solid dispersions, polymer additive is critical in pharmaceutical development. Successful ASDs utilize polymer to disperse drug molecules in order to maintain a miscible mixture and physically stable system. In a phase separated situation, drug molecules reside in the API rich domain, and their intermolecular interaction enhances the potential of molecular mobility and re-crystallization, resulting in physical instability and undesired dissolution profiles. Therefore, a well-developed ASD with enhanced API-polymer interactions may contribute to the homogeneity of the dispersion and the long-term stability of the drug product presumably by competing with API-API interaction and API-water contact.53, 54 The melted-quenched POSA-HPMCAS ASD (30 wt% DL) exhibits a surprisingly rich category of molecular interactions (Figure 7 and 8D) including O-H···O=C and O-H···F-C hydrogen bindings, and electrostatic interaction. As illustrated in Figure 7, the triazole ring of POSA is involved in two different kinds of interactions with the polymer. Although it remains an open (and interesting) question whether they exist simultaneously in a single drug molecule, we experimentally observe the following. First, both C41 and C44 in the triazole ring of POSA exhibit 13C chemical shift changes from the amorphous API reference in 1D 13C spectra (Figure 3B and C). A peak shift of HPMCAS carbonyl group is also observed. These suggest possible API-polymer contacts. Second, these two carbons exhibit intermolecular cross peaks with polymer protons in 2D

C-1H HETCOR (C41 in Figure 5C and C44 in Figure 6B), providing direct

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observations of proximity. Moreover, the 19F-13C polarization transfer experiments (Figure 8) suggest an O-H···F-C hydrogen bond between the difluorophenyl ring and the polymer. The chlorine-to-fluorine substituent in POSA represents one of the major structural differences from itraconazole. Fluorine has been found to play critical roles in target binding by forming interaction with hydrogen bond donors of polar residues or hydrophobic sidechains in proteins.49, 55 Interestingly, POSA exhibits better efficacy to a few drug-resistant fungal infections than other azoles including fluconazole, voriconazole, and itraconazole, owing to its unique chemical structure for effectively interacting with domains of the biological target.56, 57 Our findings suggest that a few active sites in the difluorophenyl and triazole rings also play critical roles in interacting with the polymer in the amorphous dispersions. ASDs with stronger intermolecular interactions are expected to be more physically stable by reducing the drug’s molecular mobility and disrupting its crystallization tendency. For example, Suryanarayanan and coworkers have utilized FT-IR spectroscopy to investigate the interaction of nifedipine (NIF) with various polymers in ASDs. Their results have suggested that NIF-PVP ASDs have the strongest interaction (of hydrogen bonding), correlating well with the findings of the longest structural relaxation times and the highest resistance to drug crystallization.11 The same relationship, i.e. the translation of stronger interaction to lower molecular mobility and enhanced physical stability, has also been identified in the study of ketoconazole and acidic excipient interactions.10 Moreover, electrostatic interactions formed by protonated molecules, despite of the low energies and poor directionality involved, have been reported to significantly enhance the physical stability of ASDs and enable a higher drug loading.6, 7 Consequently, our identified interactions explain the choice of HPMCAS as a suitable polymeric excipient for amorphous formulations of POSA from a molecule point of view. Interestingly but not surprisingly, π-stacking and electrostatic contacts in the spray dried POSA-HPMCP ASD (30 wt% DL) were identified (Figure 9D). HPMCP, different from HPMCAS, contains aromatic substitutions in the side chains (Figure 1)58, 59, which is a major contributor to the π interaction. The finding that POSA exhibits distinct interactions in the two polymer systems implies an opportunity to optimize the API-polymer contacts, in terms of the type and strength of interactions, by selecting or designing the appropriate polymers. 4.2. Molecular interactions from 2D MAS NMR Uncovering molecular details of amorphous pharmaceutical materials is challenging for several reasons. For example, amorphous materials lack long-range order. Their short-range and local structure represents a complicated system for macroscopic techniques. The relatively low drug loading of ASD formulations often results in a low population (and thus low spectroscopic intensity) of APIs. Moreover, the multicomponent nature gives compromised resolution for differentiating drug substances and excipients.


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The physiochemical environments, e.g., temperature and humidity, may also perturb the measurements.60 To investigate the molecular interaction of pharmaceutical dispersions in the solid state, various thermodynamic and spectroscopic techniques were utilized in previous studies. Our study started with the 1D spectroscopic characterization of Raman and ssNMR. Raman results did not exhibit any observable wavenumber shifts (Figure 2C), offering no sensitivity on API-polymer interaction in this particular case. Peak shifts in 1D 13C CP-MAS NMR spectra of ASDs revealed two interesting POSA chemical groups, POSA C41, and C44 respectively in Figure 3C and D, which may be involved in molecular interaction. Although the 1D

C spectra of amorphous ASDs provide preliminary information, they had limited

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resolution in providing unambiguous site-specific information. Different from the 1D strategy, 2D 13C-1H HETCOR spectra in Figure 5, 6 and 9 exhibited spatial proximity and connectivity between POSA and the two polymers, and therefore provide a more sensitive and direct measurement of molecular interactions. The spectral assignments together with the inclusion of reference spectra can significantly aid in the data interpretation of intermolecular and site-specific contacts.

Figure 10. 2D 13C-1H HETCOR spectra of 30 wt% drug-loaded POSA-HPMCAS ASD acquired at a MAS frequency of (A) 60 kHz with 1H-detection (4.7 mg samples and 14-hr acquisition time), and (B) 12 kHz with 13C-detection (86 mg samples and an acquisition time of 82-hr acquisition time). The intermolecular correlations usually present weak cross peaks in 2D HETCOR spectra of ASD due to the relatively low drug loading, nature of interactions and longer distance than intramolecular contacts. Over the past decade, new ssNMR techniques including proton detection under ultrafast MAS and DNP have remarkably advanced for the challenge of overcoming low sensitivity. For example, 60 kHz to 110 kHz MAS significantly averages one major line broadening interactions, i.e., 1H homonuclear dipolar



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coupling, and enables 1H-detected experiments for significantly improved spectral sensitivity and resolution. As a comparison illustrated in Figure 10, 1H-detected spectral quality as the

13

C-1H HETCOR (green) exhibits equally good

13

C-detected 2D spectrum (black) by using significantly less sample mass.

Technically, the high sensitivity of 1H-detected experiments can offer a better chance to probe weak intermolecular interactions. Additionally, a number of other dipolar-based 2D 1H-1H, 1H−17O, 19F-13C, 29SiC and 14N/15N−1H correlation techniques have shown great potentials for providing additional molecular

13

details about amorphous formulations.25-27, 61, 62 5. Conclusion Multiple experimental measurements5-12, 26 and molecular simulations13-15, 63 have investigated the contributing role of molecular interactions in the physical stability of amorphous formulations. Our study presents solid-state characterizations of POSA ASDs at molecular level and provides structural details of the interactions between POSA and two cellulose-based polymers, HPMCAS and HPMCP. A spectraediting and heteronuclear polarization transfer based method was utilized for unambiguous resonance assignments. Extended from 1D

13

C CP-MAS characterizations, 2D

C-1H HETCOR results have

13

successfully identified intermolecular contracts of O-H···O=C hydrogen bonding, π-π aromatic packing and electrostatic interaction. Moreover, heteronuclear

F-13C polarization transfer experiments, an

19

emerging and useful method for characterizing fluorine-containing drug substances, have suggested an interesting O-H···F-C hydrogen bonding interaction in POSA-HPMCAS ASDs. This study has successfully established a sensitive 2D ssNMR protocol for analyzing molecular interactions in solid dosages formulations. Recent progress on unambiguous resonance assignments particularly for amorphous pharmaceutical materials and technical advancements with enhanced spectral sensitivity and resolution, e.g. proton detection under ultrafast MAS as shown in this study, have exhibited a new avenue for in-depth and high-resolution investigations. Most importantly, our findings have provided valuable structural basis at a molecular level for characterizing and designing amorphous formulations. 6. Acknowledgment X. Lu is grateful to MRL Postdoctoral Research Program (Merck & Co., Inc., Kenilworth, NJ, USA). The authors thank Drs. Craig McKelvey and Michael B. Gentzler (Merck & Co., Inc., Kenilworth, NJ, USA) for insightful scientific discussions Supportive Information: additional analysis of DSC results of POSA-HPMCAS ASDs is available.


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Molecular Interactions in Posaconazole Amorphous Solid Dispersions

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