Sodium Lauryl Sulfate Competitively Interacts with HPMC-AS and

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Sodium Lauryl Sulphate Competitively Interacts with HPMC-AS and Consequently Reduces Oral Bioavailability of Posaconazole/HPMC-AS Amorphous Solid Dispersion Yuejie Chen, Shujing Wang, Shan Wang, Chengyu Liu, Ching Su, Michael Hageman, Munir Hussain, Roy Haskell, Kevin Stefanski, and Feng Qian Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00391 • Publication Date (Web): 23 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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Molecular Pharmaceutics

Sodium Lauryl Sulphate Competitively Interacts with HPMC-AS and Consequently Reduces Oral Bioavailability of Posaconazole/HPMC-AS Amorphous Solid Dispersion

Yuejie Chen1, Shujing Wang1, Shan Wang1, Chengyu Liu1, Ching Su2, Michael Hageman2, Munir Hussain3, Roy Haskell4, Kevin Stefanski2, Feng Qian1, 5

1

School of Pharmaceutical Sciences and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University, Beijing, China 2 Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Lawrenceville, New Jersey, USA 3 Drug Product Science and Technology, Bristol-Myers Squibb Company, New Brunswick, New Jersey, USA 4 Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Wallingford, Connecticut, USA 5 To whom correspondence should be addressed (Email: [email protected], Tel: 86-10-62794733)

(Manuscript for Molecular Pharmaceutics)

ACS Paragon Plus Environment


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ABSTRACT: Sodium lauryl sulphate (SLS), as an effective surfactant, is often used as a solubilizer and/or wetting agent in various dosage forms for the purpose of improving the solubility and dissolution of lipophilic, poorly water soluble drugs. This study aims to understand the impact of SLS on the solution behavior and bioavailability of HPMC-AS based posaconazole (PSZ) ASDs, and to identify the underlying mechanisms governing the optimal oral bioavailability of ASDs when surfactants such as SLS are used in combination. Fluorescence spectroscopy and optical microscopy showed that “oil-out” or “liquid-liquid phase separation (LLPS)” occurred in the supersaturated PSZ solution once drug concentration surpassed ~12 µg/mL, which caused the formation of drug rich oily droplets with initial size of ~300-400 nm. Although FT-IR study demonstrated the existence of specific interactions between PSZ and HPMC-AS in the solid state, pre-dissolved HPMC-AS was unable to delay LLPS of the supersaturated PSZ solution and PSZ-rich amorphous precipitates with ~16-18% HPMC-AS were formed within 10 min. The co-precipitated HPMC-AS was found to be able to significantly delay the crystallization of PSZ in the PSZ-rich amorphous phase from less than 10 minutes to more than 4 hours, yet co-existent SLS was able to negate this crystallization inhibition effect of HPMC-AS in the PSZ-rich amorphous precipitates and cause fast PSZ crystallization within 30 minutes. 2D-NOESY and the CMC/CAC results demonstrated that SLS could assemble around HPMC-AS and competitively interact with HPMC-AS in the solution, thus prevent HPMC-AS from acting as an effective crystallization inhibitor. In a cross-over dog PK study, this finding was found to be correlating well with the in vivo bioavailability of PSZ ASDs formulated with or without SLS. The SLS containing PSZ ASD formulation demonstrated an in vivo bioavailability ~30% of that without SLS, despite the apparently better in vitro dissolution, which only compared the dissolved drug in solution, a small fraction of the total PSZ dose. We conclude that the bioavailability of ASDs is highly dependent on the molecular interactions between drug, surfactant, and polymer, not only in the solution phase, but also in the drug-rich “oily” phase caused by supersaturation. KEYWORDS: posaconazole (PSZ), sodium lauryl sulphate, amorphous solid dispersion, molecular interaction, bioavailability


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Abstract figure:



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INTRODUCTION

Amorphous solid dispersion (ASD), where the drug disperses in polymer matrix at molecular level, is widely used in the pharmaceutical industry to improve the oral bioavailability of drugs with poor water solubility 1-6. In various ASD based oral solid dosage forms, surfactants such as sodium lauryl sulphate (SLS), Poloxamer, Tween, etc., have been used either within or outside the ASD for the purpose of improving dissolution, wetting, and ASD processing (especially, hot-melt extrusion)

7-10

. However, few investigations have been

conducted to understand their potential impacts on the supersaturated, multi-phase solution dynamics generated by ASDs. SLS is an anionic surfactant that is commonly used as a solubilizer to enhance the apparent drug solubility of lipophilic drugs, as well as a wetting agent in many dosage forms and formulation processes 8, 11, 12. Previously, we found that SLS interacted with PVP-VA to form a PVP-VA/SLS complex with lower critical aggregation concentration (CAC) and significantly increased the apparent solubility of sorafenib, a poorly water soluble drug. However, SLS negated the crystallization inhibition ability of PVP-VA against the supersaturated sorafenib in solution thus reduced the in vitro dissolution and in vivo bioavailability of the sorafenib formulation

11

. This prior experience indicated that

surfactants such as SLS could significantly impact the solution behavior and bioavailability of ASDs, through the molecular interactions with the polymeric excipients and the drug. In the current study, we aim to further explore this topic by investigating the impact of SLS on the solution behavior and in vivo bioavailability of PSZ/HPMC-AS ASDs. In fact, a HPMC-AS based ASD formulation of PSZ, Noxafil, has been approved by FDA in 2013 13. PSZ is a synthetic triazole with an extended-spectrum of activity against a range of commonly encountered fungal pathogens

14-18

. It is a BCS Class II compound with high daily dose of

300-600 mg 19, 20, low water solubility (< 2 µg/mL in FaSSIF solution), and high permeability through gastrointestinal membrane 21. PSZ is a weak base and is extremely lipophilic with a log P value great than 5, which could have contributed to its food and pH effects 21, 22. These properties made PSZ a representative model compound for nowadays new chemical entities that are currently under development across pharmaceutical industry. To understand the impact of SLS on the dissolution behavior and the in vivo


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bioavailability of PSZ ASD, we systematically examined several key events occurred in supersaturated PSZ solution with or without the presence of SLS, including the kinetics of drug supersaturation or dissolution, “oil out” or “liquid-liquid phase separation (LLPS)” induced drug-rich amorphous precipitates, the composition and crystallization kinetics of the drug-rich amorphous precipitates, etc. It’s worth noting that the drug-rich amorphous precipitates induced by LLSP have been hypothesized as potential reservoirs of high energy drug to maintain enhanced and prolonged drug dissolution

23, 24

. In this study, we confirmed

that substantial amount of HPMC-AS co-precipitated with PSZ during LLPS, and served as crystallization inhibitor for the amorphous drug. At the same time, we also revealed the molecular interaction mechanisms between PSZ, HPMC-AS, and SLS, by FT-IR, 2D-NOESY, and CMC/CAC measurements. SLS was found to interact with the hydrophobic moieties on HPMC-AS competitively with PSZ, thus prevent HPMC-AS from effectively inhibit PSZ crystallization of amorphous and subsequently induced the depletion of the amorphous drug reservoir. This phenomenon was proven to be in vivo relevant in a cross-over dog PK study where the bioavailability of HPMC-AS based PSZ ASD formulations with or without SLS was compared. The SLS containing formulation showed a significantly reduced bioavailability of ~30% relative to SLS-free ASD formulation.

MATEARIALS AND METHODS Materials

PSZ was purchased from Beijing Ouhe Technology Co. Ltd., (Beijing, China). HPMC-AS was provided by Shin-Etsu Chemical Co., Ltd (Tokyo, Japan). Sodium lauryl sulphate (SLS) (Kolliphor® SLS Fine) was provided by BASF Chemical Company Ltd. (Ludwigshafen, Germany). All buffer salts used for dissolution medium, as well as methanol and tetrahydrofuran (THF) used for sprays drying were obtained from Beijing Chemical Works (Beijing, China). The chemical structure and key physiochemical properties of PSZ, HPMC-AS, and SLS are summarized in Figure 1. Measurement of the aqueous solubility of crystalline PSZ



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Aqueous solubility of crystalline PSZ in FaSSIF solution in presence of either HPMC-AS or SLS, was determined by stirring excess amount of crystalline drug in the dissolution medium, followed by sonication (10 min), and then shaking using an orbital shaker (37°C, Burrell wrist action shaker, model 75) for 24 hours. The suspension was subsequently centrifuged twice at 13,000 rpm (Thermo Scientific Sorvall Legend Micro 21R; Thermo Fisher Scientific, Waltham, USA) for 30 min, and the drug concentration in the supernatant was determined by HPLC/UV-vis. The concentration of HPMC-AS and/or SLS is 0.3, 1, or 3 mg/mL. Determine the degree of supersaturation of PSZ solution

During the dissolution of ASD, drug may exist in supersaturation state in the dissolution medium. The degree of supersaturation can be calculated using the following equation 25: ஼

ܵ=஼

(1)

೐೜

Where S is the supersaturation ratio, C is the solution drug concentration, ‫ܥ‬௘௤ is the equilibrium solubility of the crystalline drug. In theory, a drug solution with higher degree of supersaturation has higher propensity to lower its free energy by amorphous phase separation or drug crystallization. Preparation of the spray-dried ASDs and ASD-based tablets

PSZ/HPMC-AS ASDs with 25% drug loading were prepared by spray drying a 3 wt% solution in THF/ methanol (1:1 w/w ratio) co-solvent using a Yamato spray dryer (ADL 311S, Yamato Scientific Co., Ltd., Tokyo, Japan). The solution flow rate was 8 mL/min, inlet temperature was 80 °C, outlet temperature was ~45 °C, and atomizing N2 pressure was 0.1 MPa. The spray-dried ASDs were vacuum dried at room temperature for at least 24 hours to remove any residual solvents before further use. The solid dispersions were confirmed to be amorphous by PXRD and differential scanning calorimetry (DSC) (data not shown). Two types of PSZ tablets were prepared according to composition listed in Table 1. Firstly, physical blends of ASD and different excipients were prepared by sieving their mixtures through 0.3 mm sieve until a homogeneous mixture was obtained, and the obtained powder mixtures were compressed using a lab press (Carver Press, C-NE 3888, USA) to


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produce PSZ tablets for in vivo dog PK studies. The hardness of the tablets were 4-6 Kgf, measured by a tablet hardness tester (YD-1A, Tianjin Tuopu Instrument Co. Ltd., Tianjin, China). Fourier transform infrared spectroscopy (FT-IR) study of ASDs

To study the molecular interaction between PSZ and HPMC-AS in solid state, FT-IR spectra of amorphous PSZ, HPMC-AS, and different ASDs (with 20%, 40%, 60%, and 80% drug loading) were collected by a Fourier transform infrared spectroscopy (Vertex 70, Bruker Optics, Ettlingen, Germany) with a spectral resolution of 4 cm-1. The IR spectra between 4000–600 cm-1 were recorded for comparison. Effect of different excipients on the precipitation kinetics of supersaturated PSZ solution

In order to assess the abilities of HPMC-AS and SLS to maintain the solution supersaturation of PSZ, HPMC-AS and SLS were pre-dissolved in Fasted State Simulated Intestinal Fluid (FaSSIF, prepared according to literature 6) at 0.3 mg/mL, 1 mg/mL, or 3 mg/mL. PSZ solution in THF was prepared at 20 mg/mL. In each 5 mL of FaSSIF pre-dissolved with various excipients, 50 µL of PSZ solution was added. The solution was then vibrated at a frequency of 100 RPM using a shaker (37 °C, Burrell wrist action shaker, Model 75). After 20 min, 1, 2 and 4 hours, 0.3 mL of solution was withdrawn and centrifuged at 13,000 RPM for 3 min. The obtained clear supernatant was analyzed for drug concentration. Meanwhile, the polymer concentration within the precipitate was determined using a HPLC/ELSD method as we developed and detailed earlier 26. Analyze the phase separation of supersaturated PSZ solutions and self-assembly of SLS/HPMC-AS by a fluorescence measurement

A fluorescence probe, pyrene, was used to investigate the phase separation of supersaturated PSZ solution. As used and reported earlier

11, 27, 28

, pyrene is an

environment-sensitive, hydrophobic fluorescence probe, with two characteristic emission peaks (I1 at 373 nm, I3 at 383 nm) that give rise to different intensities at environments with different hydrophobicity. Therefore, the change of peak ratio of I1 and I2 indicates solution phase change. Fluorescence spectra were collected using a Fluoromax-3 (Horiba Jobin Yvon,



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Edison, NJ, USA) spectrometer. The excitation wavelength was 332 nm, and the emission spectrum was collected at 1.16 nm intervals from 350 to 420 nm. The dissolution medium used in this study was 50 mM PBS buffer (pH=6.5), and the fluorescence spectra were collected within 5 min after the solution preparation. This method was also used to study the self-assembly of SLS in presence of PVP-VA, similar as we reported before 11. The critical micelle concentration (CMC) of SLS, or critical aggregation concentration (CAC) of HPMC-AS/SLS combination, was identified at the concentration where the two peak (I1 at 373 nm, I3 at 383 nm) intensity ratio starts to decrease. Powder x-ray diffraction (PXRD) analysis

PXRD was used to investigate the crystallinity of PSZ in the PSZ-rich amorphous precipitates. PXRD patterns were collected on a PANalytical X’ pert Powder X-Ray Diffractometer (copper X-ray tube, 40 kV * 40 mA, Almedo, The Netherlands). Samples were placed on the zero-background silicon sample holder. The PXRD experiments was performed with automatic divergence slit graphite monochromator, 0.2 mm receiving slit, and continuous scanned from 5° to 45° (2θ) at a speed of 4°/min. NMR analysis of the specific interactions between HPMC-AS and SLS

The molecular interactions between HPMC-AS and SLS in aqueous environment were characterized by two-dimensional nuclear Overhauser effect spectroscopy (2D-NOESY). HPMC-AS, SLS, and HPMC-AS/SLS (1:1 w/w) combination were dissolved in 70 mM PBS buffer (prepared in D2O, with pH maintained at 6.5) at the concentration of 15 mg/mL, and their 1H NMR and 2D-NOESY spectra were acquired at 310 K using a Bruker AV-400 (Bruker BioSpin GmbH, Rheinstetten, Germany) NMR operating at 400 MHz. For 2D-NOESY, the mixing time was set to 0.1 s (for HPMC-AS and HPMC-AS/SLS system) or 0.5 s (for SLS), and the experiments were performed with a 2 s relaxation delay and 0.32 s acquisition time. In vitro dissolution study under non-sink condition The effect of HPMC-AS or SLS on the dissolution behavior of PSZ ASD were


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investigated in a non-sink dissolution study. For each set of experiment, a total of 15 samples (total 5 time points with 3 replicate) weighted 31.5 mg (5 mg API blended with different excipients, refer to Table 1) were prepared. Each sample was loaded into 50 mL centrifuge tube followed by addition of 10 mL FaSSIF solution. The samples were vortexed for 30 s prior to being placed on a shaker at 100 RPM, with temperature maintained at 37 °C. Dissolution test for each set of samples was terminated after 10 min, 0.5, 1, 2, and 4 hours, followed by centrifuging the samples at 13000 RPM for 3 min (Avanti J-26S XP; Beckman Coulter, Brea, CA, USA). The supernatant was analyzed for drug concentration, and the precipitate was analyzed for crystallinity by PXRD experiment. In vivo dog PK study of PSZ ASD tablets with and without SLS The in vivo PK performance of two differently formulated PSZ tablets (100 mg API blended with different excipients, refer to Table 1) was evaluated in male beagle dogs (∼10 kg) using a crossover study (n=4). Dogs were fasted overnight with no intake of water 1 hour before and after dosing. Drug tablet administration was followed immediately by gavage with 20 mL water. Blood samples (2 mL) were withdrawn from the cephalic vein pre-dosing, and at 10 min, 0.5, 1, 2, 4, 6, 8, 24 and 48 h after dosing. The samples were then placed in EDTA-containing vacutainer blood collection tubes. The samples were subsequently centrifuged at 400 × g for 15 min at 4°C, and the plasma was isolated for LC/MS analysis (PE Applied Biosystems, Foster City, CA, USA).

RESULTS AND DISCUSSION Solubility of crystalline PSZ in the presence of HPMC-AS and/or SLS

The aqueous solubility of crystalline PSZ in the presence of HPMC-AS and/or SLS was summarized in Table 2. Without HPMC-AS or SLS, the aqueous solubility of crystalline PSZ in FaSSIF is very low at 1.7 µg/mL. With the addition of up to 3 mg/mL HPMC-AS, PSZ solubility remains unchanged, indicating that HPMC-AS is not able to increase the solubility of crystalline PSZ. Surfactants are routinely employed to increase the apparent aqueous solubility of poorly



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water soluble drugs. Above the CMC, drug could be encapsulated within the hydrophobic core of the micelles, which usually lead to significantly increase in the apparent solubility of lipophilic drugs. As shown in Table 2, the solubility of PSZ increased to 2.2 µg/mL with 0.3 mg/mL SLS, and to 31.2 µg/mL (a ~18.5 fold increase) with 3 mg/mL SLS. Combination of 0.3 mg/mL HPMC-AS and 3 mg/mL SLS solubilized PSZ synergistically, where PSZ solubility increased ~ 21.4 times to 36.4 µg/mL. Further increasing HPMC-AS to 3 mg/mL while maintaining SLS at 3 mg/mL only increased the solubility of PSZ slightly to 38.5 µg/mL. Molecular interaction between PSZ and HPMC-AS in the solid state

To investigate the molecular interaction mechanism between PSZ and HPMC-AS, FT-IR spectra of pure amorphous PSZ, HPMC-AS, as well as PSZ/HPMC-AS ASDs with 20, 40, 60, and 80% drug loading, were collected and compared in Figure 2. The two peaks centered at 1702 cm-1 and 1691 cm-1 were respectively assigned to the free carbonyl of amorphous PSZ, and the same moiety when inter-molecular hydrogen bonding occurs between the PSZ molecules. The peak at 1654 cm-1 was attributed to the imine group of amorphous PSZ. In the PSZ/HPMC-AS ASD systems, with the increasing of HPMC-AS content up to 60%, the PSZ carbonyl peak gradually blue-shifted from 1702 cm-1 to 1707 cm-1, indicating increased extent of PSZ/HPMC-AS molecular interaction. It’s worth noting that H-bonding typically causes a red shift of the carbonyl. It’s possible that this PSZ carbonyl blue shift was caused by the approaching of some electron rich groups on HPMC-AS, which strengthened the bond energy. Meanwhile, the peak centered at 1691 cm-1 disappeared with the addition of HPMC-AS, indicating the disruption of PSZ-PSZ interaction by HPMC-AS. The peak at 1654 cm-1 also disappeared with more than 20% HPMC-AS, indicating the formation of specific interaction on the imine group of PSZ and HPMC-AS. The peak at 1620 cm-1 is assigned to phenyl group of amorphous PSZ. No peak shift was observed, and the peak intensity gradually decreases with the decrease of PSZ, indicating the phenyl groups are not involved in the drug-polymer specific interactions. These specific interactions between PSZ and HPMC-AS could have contributed to the strong crystallization inhibition effect of HPMC-AS towards amorphous PSZ, which will be discussed later.


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Supersaturation of PSZ in the presence of HPMC-AS or SLS

Dissolution of amorphous drug could produce supersaturation, which could generate higher in vivo bioavailability if the supersaturation was prolonged by an optimally designed formulation. Therefore, it’s necessary to evaluate the formulation’s ability in maintaining drug supersaturation in aqueous solution. In this study, we investigated the kinetics of PSZ supersaturation in the presence of pre-dissolved HPMC-AS or SLS at the concentration of 0.3, 1, or 3 mg/mL, and the amorphous precipitates from solution containing 1 mg/ml and 3 mg/ml HPMC-AS were also collected and analyzed for polymer concentration. As shown in Figure 3, without HPMC-AS or SLS, the concentration of PSZ in FaSSIF decreased rapidly within 20 min from the initial 200 µg/mL to ~20 µg/mL caused by LLPS. HPMC-AS at the concentrations of 0.3, 1, or 3 mg/mL did not change the phase separation kinetics of the supersaturated PSZ solution, which still rapidly decreased to ~25 µg/mL after 20 min and further decreased to ~ 10 µg/mL over the following 4 hours (Figure 3A). With a simple calculation using solution drug concentration and volume of dissolution medium, we estimated that >90% drug precipitated from the solution. Note that PSZ solution concentration in some measurements exceeded the LLPS-trigging concentration of ~12 µg/mL. This could be caused by experimental artifacts since some tiny drug rich droplets were difficult to be completely removed by centrifugation. Therefore, the measured PSZ concentration should be termed as “apparent drug concentration”, as we indicated in the figures. With 0.3 mg/mL SLS, LLPS occurred rapidly and PSZ solution concentration decreased similarly as the solution without SLS. Increasing the SLS concentration to 1 mg/mL did not prevent the occurrence of rapid LLPS yet generated a modestly higher drug concentration between 20 min to 2 hours. Only when the SLS concentration was further increased to 3 mg/mL, precipitation of highly supersaturated PSZ could be effectively prevented and the drug concentration was maintained at ~170 µg/mL over 4 hours (Figure 3B). This is not surprising because the solubility of crystal PSZ in FaSSIF with 3 mg/mL SLS is ~ 31.2 µg/mL, much higher than that in blank FaSSIF solution (~ 1.7 µg/mL) (Table 2), therefore, the supersaturation ratio of PSZ in FaSSIF with 3 mg/mL SLS (~ 5.3) is much lower than that in blank FaSSIF (~30-70). It’s worth noting that, 3 mg/mL SLS concentration is too high to



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achieve in vivo despite its ability to keep PSZ in solution. Assuming 250 mL gastric fluid volume, 3 mg/mL SLS translates into 750 mg SLS, an amount far exceeds safety allowed value. While with practically viable amount (such as 0.3 mg/mL), SLS has no effect to maintain the supersaturation of PSZ. LLPS in supersaturated PSZ solution with or without the presence of HPMC-AS or SLS

During process development of API (active pharmaceutical ingredient) crystallization, it has been commonly observed that a highly supersaturated drug solution could undergo LLPS or “oil-out” to reduce its free energy, if supersaturation did not induce immediate crystallization 28, 29. LLPS could also be induced by the dissolution of ASDs, and the drug-rich oily phase or amorphous precipitates were hypothesized to be able to provide a drug reservoir to keep supplying high energy drugs to enhance dissolution and bioavailability, although this hypothesis has yet been confirmed in vivo 23. Adding 0.2 mL PSZ solution (20 mg/mL in THF) into 20 mL PBS buffer, we observed the occurrence of LLPS in the supersaturated PSZ solution under optical microscope, initially in the form of spherical and transparent oily droplets with diameters of ~300-500 nm (data not shown). To quantitatively determine the concentration threshold that triggers LLPS, an environmentally sensitive probe, pyrene was included in the solution and the ratio of the intensities of I1 (373 nm) and I3 (383 nm) peaks was determined (Figure 4). In the absence of HPMC-AS, the I1 /I3 ratio remained constant (~ 1.5) when the PSZ concentration was under ~12 µg/mL, indicating that the probe was uniformly dispersed in a hydrophilic environment. However, once the PSZ concentration exceeded 12 µg/mL, the I1 /I3 ratio gradually decreased with the increasing of drug concentration, indicating the formation of a more hydrophobic phase in the supersaturated PSZ solution. We also observed that the presence of HPMC-AS and SLS did not change the ~12 µg/mL LLPS onset concentration, which is consistent with the findings in Figure 3. Crystallization kinetics of amorphous precipitates with and without HPMC-AS

The phase separated PSZ solution, with and without the presence of HPMC-AS, was centrifuged (13,000 RPM, 3 min) and the amorphous precipitates were collected and


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subjected to PXRD analysis for their crystallization kinetics. Without HPMC-AS, the precipitates were shown to be amorphous initially by the broad and featureless PXRD pattern. However, crystalline PSZ peaks were detected after 10 min, suggesting the high crystallization tendency of amorphous PSZ within the oily/amorphous phase. In contrast, with the presence of 1 mg/mL HPMC-AS in the solution, the precipitates remained crystal-free by PXRD analysis for at least 1 hour (Figure 5), suggesting effective inhibition of PSZ crystallization. To understand the underlying mechanism, the precipitates were collected, dried, and analyzed by HPLC/ELSD to determine the PSZ and HPMC-AS content, as listed in Table 3. To our surprise, although the amorphous precipitates were PSZ rich, they also

contained ~16 wt% of HPMC-AS, when the solution HPMC-AS concentration was 1 mg/mL. Further increasing the HPMC-AS concentration to 3 mg/mL only slightly increased its content in the precipitates to 18%. We hypothesized that the co-precipitated HPMC-AS was essential to inhibit PSZ recrystallization within the amorphous precipitates, which was further validated in the following sections. Molecular interaction mechanisms between HPMC-AS and SLS

SLS was reported to be able to assemble around certain type of polymers through specific interactions

11, 30

. Here we studied the self-assembling behavior of SLS with or

without the presence of HPMC-AS, and also applied 2D-NOESY analysis to investigate the interaction mechanisms between HPMC-AS and SLS. a) Assembling behavior of SLS and SLS/HPMC-AS Systems

We used the environment-sensitive dye, pyrene, to study the self-assembling behavior of SLS and SLS/HPMC-AS, similar as we reported previously 11. The fluorescence peak ratio I1 (373 nm)/I3 (383 nm) was plotted against the apparent SLS concentration, and CMC/CAC values of the SLS, SLS/HPMC-AS systems were identified as the bending points of the I1/I3 ratio (Figure 6). According to Figure 6, SLS has a CMC value of ~ 8.0 mM, while addition of 3 mg/mL HPMC-AS allowed SLS to assemble at a much lower concentration of ~ 4.2 mM. These results suggest that SLS molecules interact with HPMC-AS and assemble around HPMC-AS at a lower concentration.



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b) 2D-NOESY

2D-NOESY measurement provides information on the distance between protons that are spatially close to each other, thus has been used to study the intramolecular or intermolecular interaction in solutions

31, 32

. We employed this technique here to investigate the interaction

between HPMC-AS and SLS in aqueous solution. The 2D-NOESY spectra of HPMC-AS (15 mg/mL), SLS (15 mg/mL), and HPMC-AS/SLS combination (both are 15mg/mL) are shown in Figure 7. The peak of each proton of HPMC-AS and SLS were assigned according to literature 33, 34. There are three types of cross peaks colored with yellow, red, and blue, which showed the cross peaks between protons of HPMC-AS/SLS combination, HPMC-AS, and SLS, respectively. For HPMC-AS/SLS systems, strong NOE interactions between the protons on hydroxypropyl group and the cellulose chain (3.48-3.63 ppm) of HPMC-AS, and the protons on hydrophobic tails (1.36 ppm for 1-H and 0.95 ppm for 2-H) of SLS are observed, indicating close proximity of the hydrophobic tail of SLS to the hydroxypropyl groups and cellulose chains of HPMC-AS. In another word, hydrophobic interactions formed between HPMC-AS and SLS. As we discussed earlier, the molecular interaction between HPMC-AS and PSZ serves to inhibit the crystallization of amorphous PSZ in the precipitates. The fact that SLS interacts with HPMC-AS in a competitively manner might disrupt the interaction between PSZ and HPMC-AS, thus to jeopardize the ability of HPMC-AS to inhibit PSZ crystallization. PSZ/HPMC-AS ASD solution behavior under non-sink condition with or without SLS Figure 8 showed the in vitro dissolution results of two PSZ/HPMC-AS based ASD

formulations with and without SLS (Table 1) under non-sink condition. It must be pointed out that the amount of solution PSZ only accounted for a small fraction of the total drug dose (~10% in maximal, as indicated by the “drug release %”) and the highest drug concentration achieved by either formulation was less than 80 µg/mL. Comparing the solution drug kinetics, formulation II (with external SLS) demonstrated superior dissolution result than formulation I (without SLS), likely due to the increased solubility of PSZ and increased wetting of the formulation with the presence of SLS (Table 2). Since the majority (~90%) of PSZ was found in the amorphous precipitates induced by


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LLPS, we collected the PSZ-rich precipitates over 4 hour and analyzed them by PXRD. Shown on Figure 9A, with extra HPMC-AS while no SLS, PSZ-rich precipitates remained amorphous throughout the 4 hour dissolution time, while when SLS was introduced into the formulation, drug crystallization was detected in the PSZ-rich precipitates within 1 hour (Figure 9B). These observation is consistent with our earlier conclusion that HPMC-AS inhibit PSZ crystallization in the precipitated amorphous state, while SLS competitively interacts with HPMC-AS thus prevent HPMC-AS from effectively inhibit PSZ crystallization. Therefore, although demonstrated an apparently inferior dissolution performance (Figure 8), formulation I was about to maintain much more drug in molecule state, if both solution drug and precipitated drug were both taken into consideration. Dog PK comparison of the PSZ ASD based tablet formulations with or without SLS

To confirm the in vivo relevance of all above findings, we compared the in vivo performance of the two PSZ ASD formulations with or without SLS in a cross-over dog PK study (Figure 10). Strikingly, Formulation I (SLS-free) showed a significantly higher bioavailability, with Cmax and AUC of about 2.5 and 3.4 folds higher than that of formulation II (formulated with SLS). The in vivo results clearly demonstrated that the in vivo performance of the ASD based formulations was better correlated with the fate of the entire dose of the drug, including both the solution drug and the precipitated drug. Also, the interplays between drug, polymer and surfactant could critically affect each process occurring in the solution and the ultimate bioavailability. Findings in this work also further reinforced our earlier observations and conclusions 2, 11.

CONCLUSION

In short, we conclude that 1). Amorphous oil-droplets or precipitates, induced by LLPS or “oil-out” during the dissolution of ASDs, could serve as “high energy drug reservoirs” to enhance drug bioavailability; 2). Polymers could co-precipitate with amorphous drugs and serve as crystallization inhibitors to preserve the drug reservoir; 3). Surfactants such as SLS could have competitive molecular interactions with the polymer, thus might negate the



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crystallization inhibition of the polymer against the amorphous drug; 4). The above findings regarding the solution behavior of ASDs have clear in vivo correlation.

ACKNOWLEDGMENTS

This research is supported by China National Nature Science Foundation (project number 81573355), and Bristol-Myers Squibb Company (Lawrenceville, NJ, USA). FQ also thank the start-up funds provided by the Center for Life Sciences at Tsinghua and Peking Universities (Beijing, China), and by the China Recruitment Program of Global Experts.


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Achieved

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Equilibrium State at Supersaturated Drug

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Table 1. Composition of the PSZ/HPMC-AS ASD based tablet formulations for in vitro dissolution study and in vivo dog PK study.

Contents ASD

Excipients

Weight

Formulation I

Formulation II

PSZ

15.9 % (100 mg)

15.9 % (100 mg)

HPMC-AS

47.6 % (300 mg)

47.6 % (300 mg)

Colloidal Silicon Dioxide

1.6 % (10 mg)

1.6 % (10 mg)

CMCNa

7.9 % (50 mg)

7.9 % (50 mg)

MCC PH302

15.9 % (100 mg)

15.9 % (100 mg)

HPMC-AS

11.1 % (70 mg)

-

SLS

-

11.1 % (70 mg)

100% (630 mg)

100% (630 mg)


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Table 2. Solubility of crystalline PSZ in FaSSIF (pH 6.5) with the presence of HPMC-AS and/or SLS with different concentrations. (n=3 for all experiments)

Solvent

Solubility (µg/mL)

FaSSIF

1.7 (± 0.1)

0.3 mg/mL HPMC-AS

1.7 (± 0.1)

1 mg/mL HPMC-AS

1.7 (± 0.1)

3 mg/mL HPMC-AS

1.7 (± 0.1)

0.3 mg/mL SLS

2.2 (± 0.1)

1 mg/mL SLS

5.1 (± 0.1)

3 mg/mL SLS

31.2 (± 0.3)

0.3 mg/ml HPMC-AS + 3 mg/ml SLS

36.4 (± 0.6)

3 mg/ml HPMC-AS + 3 mg/ml SLS

38.5 (± 0.4)



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Table 3. Amount of HPMC-AS within the drug rich amorphous precipitates. (n=3 for all experiments, the amount of HPMC-AS was determined by a HPLC/ELSD method)

Amount of HPMC-AS in solution

Amount of HPMC-AS in precipitates (wt%)

1 mg/mL

16% (± 2%)

3 mg/mL

18% (± 3%)


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Figure 1. Chemical structure of posaconazole (PSZ), hydroxypropylmethylcellulose acetate succinate (HPMC-AS), and sodium lauryl sulphate (SLS).



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Figure 2. FT-IR spectra of pure amorphous PSZ, HPMC-AS, and PSZ/HPMC-AS ASD with different drug/polymer ratios (P: PSZ; H: HPMC-AS). On the spectrum of PSZ, the two carbonyl peaks centered at 1702 cm-1 and 1691 cm-1 correspond to the free carbonyl groups, and the carbonyl groups of PSZ molecules that formed inter-molecular hydrogen bonding. The peak at 1654 cm-1 was attributed to the imine group of amorphous PSZ.


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Figure 3. Supersaturation of PSZ solution in the presence of different amount of (A) HPMC-AS, or (B) SLS. The initial drug concentration was 200 µg/mL. Note: The 200 µg/mL concentration is not an experimentally measured value but represents the theoretical starting concentration calculated based on the amount of PSZ added. (n=3 for all experiments)



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Figure 4. Ratio of fluorescence intensity between the first (I1 = 373 nm) and the third peak (I3 = 383 nm) of pyrene emission spectra as a function of PSZ concentration with or without the presence of HPMC-AS or SLS. The arrow indicated the onset of LLPS concentration, where a sharp decrease in I1 /I3 ratios occurred. (n=3 for all experiments)


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Figure 5. PXRD patterns of (from top to bottom) crystal posaconazole (PSZ); PSZ-rich precipitate collected after one hour following the addition of PSZ into FaSSIF containing 1 mg/mL HPMC-AS; 10 min after the addition of PSZ into FaSSIF solution without HPMC-AS, and immediately after the addition of PSZ into FaSSIF solution without HPMC-AS.



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Figure 6. Change of fluorescence intensity ratio (I1/I3) of pyrene as a function of total SLS concentration in solutions containing SLS and HPMC-AS/SLS. The bending points of the I1/I3 ratio were identified as the CMC or CAC values of the SLS and SLS/HPMC-AS solutions, respectively.


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Figure 7. Overlay of the 2D 1H-1H NOESY spectra of HPMC-AS/SLS combination (yellow), HPMC-AS (red) and SLS (blue). Regions corresponding to the cross peaks from interaction between HPMC-AS and SLS are zoom viewed in boxes, with the chemical shifts labelled aside.



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Figure 8. In vitro dissolution of the PSZ/HPMC-AS ASD based formulations with (Formulation II) or without SLS (Formulation I) (n=3). The composition of the two formulations was listed in Table 1.


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Figure 9. PXRD patterns of the PSZ-rich precipitates generated by dissolving two PSZ/HPMC-AS ASD based formulations with (Formulation II) or without (Formulation I) SLS. The compositions of the two formulations are listed in Table 1.



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Figure 10. In vivo dog PK profiles of two PSZ/HPMC-AS ASD based formulations with (Formulation II) or without (Formulation I) SLS (n=4).


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Sodium Lauryl Sulfate Competitively Interacts with HPMC-AS and

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