Improving the drug load and in vitro performance of supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS) using polymeric precipitation inhibitors

Journal Pre-proofs Improving the Drug Load and in vitro Performance of Supersaturated SelfNanoemulsifying Drug Delivery Systems (Super-SNEDDS) using Polymeric Precipitation Inhibitors J. Bannow, Y. Yorulmaz, K. Löbmann, A. Müllertz, T. Rades PII: DOI: Reference:

S0378-5173(19)31005-1 https://doi.org/10.1016/j.ijpharm.2019.118960 IJP 118960

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

25 October 2019 11 December 2019 12 December 2019

Please cite this article as: J. Bannow, Y. Yorulmaz, K. Löbmann, A. Müllertz, T. Rades, Improving the Drug Load and in vitro Performance of Supersaturated Self-Nanoemulsifying Drug Delivery Systems (SuperSNEDDS) using Polymeric Precipitation Inhibitors, International Journal of Pharmaceutics (2019), doi: https:// doi.org/10.1016/j.ijpharm.2019.118960

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Improving the Drug Load and in vitro Performance of Supersaturated Self-Nanoemulsifying Drug Delivery Systems (Super-SNEDDS) using Polymeric Precipitation Inhibitors J. Bannow1, Y. Yorulmaz1, K. Löbmann1, A. Müllertz1, T. Rades1,2* 1University

of Copenhagen, Department of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen,

Denmark 2Åbo

Akademi University, Faculty of Science and Engineering, Tykistökatu 6A, FI-20521, Turku, Finland *Corresponding author: [email protected]

ABSTRACT In this study, the influence of the polymeric precipitation inhibitor (PPI) PVP/VA 64 (polyvinylpyrrolidone-co-vinyl acetate) on the physical stability and in vitro performance of supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS) containing the model drug simvastatin (SIM) was investigated. A heating-cooling cycle was employed to dissolve (i) the drug in the SNEDDS preconcentrate, generating super-SNEDDS, or (ii) the drug and PPI generating PPI super-SNEDDS, both containing drug loads of 200 % and 250 % (with regard to the equilibrium solubility of SIM in the blank SNEDDS). PPI super-SNEDDS were prepared at PPI concentrations

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of 1 %, 10 % and 20 % (w/w), respectively. The formulations were characterized using polarized light microscopy, dynamic light scattering, rheological profiling and dynamic in vitro lipolysis. The physical stability of PPI super-SNEDDS correlated with an increase in viscosity due to the additionally dissolved PVP/VA 64. PPI super-SNEDDS with drug loads of 200 % and 250% containing 20 % (w/w) PPI showed no drug recrystallization after more than 6 months of storage at room temperature, whereas PPI-free super-SNEDDS (250 % drug load) recrystallized within two hours after equilibration to room temperature. All formulations formed nanosized droplets after emulsification in Milli-Q water. The droplet size was not affected by the PPI, but increased slightly with increasing drug load (z-average of 47.3 ± 0.4 nm for SNEDDS with 200 % drug load and 55.6 ± 1.3 nm for SNEDDS with 250 % drug load). PPI super-SNEDDS with a drug load of 200 % containing 20 % (w/w) PVP/VA 64 showed an improved performance during dynamic in vitro lipolysis, maintaining a 2.5-fold higher degree of supersaturation after 15 min of digestion compared to PPI-free super-SNEDDS of the same drug load. In conclusion, the study demonstrated the feasibility of stabilizing higher drug loads and improving the in vitro performance of superSNEDDS by incorporating PVP/VA 64 into the preconcentrate.

KEYWORDS: Lipid based drug delivery systems (LbDDS), Self-nanoemulsiying drug delivery system (SNEDDS), supersaturation, polymeric precipitation inhibitors (PPIs), supersaturatedSNEDDS (super-SNEDDS)

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1.

INTRODUCTION

The use of combinatorial chemistry and high throughput screening in the pharmaceutical industry has resulted in a continuously increasing amount of small organic drug candidates that exhibit poor aqueous solubility resulting in limited oral bioavailability (Lipinski et al., 2012). Over the past decades, various enabling formulation strategies have been developed to overcome this issue, including the use of polymer-based and lipid-based oral formulations (Siepmann et al., 2019). The production of amorphous solid dispersions (ASDs), where the drug and polymeric excipient form a homogenous blend, is the most established and commercially successful technique to improve the oral bioavailability of poorly soluble drugs (Baghel et al., 2016; Van den Mooter, 2012). The polymer not only stabilizes the thermodynamically unstable amorphous drug during storage, but can also act as a polymeric precipitation inhibitor (PPI), maintaining a potentially supersaturated state in the gastrointestinal tract (GIT) by preventing precipitation of the drug after dissolution, an effect which is often referred to as “parachute effect” (Augustijns and Brewster, 2012; Guzmán et al., 2007). Since supersaturated drug concentrations in the GIT increase the driving force for absorption, preservation of the supersaturated state is crucial for the bioavailability improvement of poorly water-soluble drugs (Sun and Lee, 2013). The apparent degree of supersaturation (aDS) (Blaabjerg et al., 2018) can be calculated by dividing the concentration of the drug in a supersaturated solution (csupersaturation) by the concentration of the drug in a saturated solution (cequilibrium). aDS = csupersaturation / cequilibrium

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The mechanisms of polymeric precipitation inhibitors (PPIs), such as cellulose ethers (hydroxypropylmethylcellulose (HPMC), HPMC acetate-succinate (HPMC-AS) etc.) or vinyl polymers (polyvinylpyrrolidone (PVP), (polyvinylpyrrolidone-co-vinyl acetate (PVP/VA) etc.) to

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stabilize supersaturated drug concentrations include the inhibition of nucleation and crystal growth (Warren et al., 2010). In addition, some of these PPIs are known to increase the apparent solubility of the drug and thereby lowering the thermodynamic driving force for both processes (Loftsson et al., 1996). Especially high PPI concentrations in solution can lead to a substantial increase in viscosity and result in a kinetic stabilization of the supersaturated state by lowering the diffusivity of drug molecules (Miller et al., 2008). Moreover, PPIs can also directly interact with the crystalmedium interface and thereby affect the integration of molecules into the crystal lattice (Brouwers et al., 2009). Consequently, the inhibitory effects of PPIs remain strongly dependent on the drug/PPI combination and thus are difficult to predict (Laitinen et al., 2017). Another formulation strategy to improve the oral delivery of poorly water-soluble drugs that has received considerable attention over the last years is the use of lipid-based drug delivery systems (LbDDS) (Pouton and Porter, 2008). In LbDDS, the drug is usually dissolved in the preconcentrate and therefore bypasses the potentially absorption-limiting dissolution step for poorly water-soluble drugs in the GIT, consequently resulting in an improved oral bioavailability (Fatouros and Mullertz, 2008). The composition of LbDDS ranges from simple solutions of the drug in an oily carrier to more complex self-nanoemulsifying drug delivery systems (SNEDDS) that are multicomponent mixtures of lipids, emulsifiers and co-solvents forming an isotropic preconcentrate (Müllertz et al., 2010). After administration, lipids are digested by gastrointestinal lipases and form mixed micelles containing digestion products (fatty acids and monoglycerides) and endogenous components (bile salts, phospholipids and cholesterol) that interact with the drug molecules and thereby influence drug solubilization and absorption (Porter et al., 2007). In particular, SNEDDS undergo fast digestion due to the large surface area created after the formation of nanosized emulsion droplets.

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None withstanding this, multiple studies demonstrated the ability of SNEDDS to improve the bioavailability of poorly water-soluble drugs (Attivi et al., 2010; Cui et al., 2009; Kang et al., 2004; Nielsen et al., 2008). The usual drug load applied in SNEDDS ranges from 50-90 % of the drug’s equilibrium solubility (Seq) in the preconcentrate (conventional SNEDDS) and often results in large amounts of formulation needed to reach therapeutic doses (Larsen et al., 2013). To overcome this limitation, Thomas et al. (2012) developed supersaturated SNEDDS (super-SNEDDS) containing drug loads well above the Seq in the preconcentrate (Thomas et al., 2013; Thomas et al., 2012a). In vitro and in vivo evaluations demonstrated the ability of super-SNEDDS containing simvastatin (SIM) and halofantrine to show equal or improved performance compared to conventional SNEDDS. Supersaturated drug concentrations have also been observed after emulsification of SIM loaded super-SNEDDS during in vitro lipolysis, which was followed by amorphous precipitation of the drug. It is assumed that the interplay of amorphous precipitate and supersaturated drug concentrations led to a considerably higher bioavailability of SIM in beagle dogs compared to the dosing of multiple units of conventional SNEDDS (Thomas et al., 2013). In an attempt to maintain supersaturation after dispersion, Gao et al. (2003) developed supersaturable SEDDS (S-SEDDS) by adding PPIs to conventional SNEDDS. A prolonged supersaturation after dispersion in aqueous media and a fivefold higher oral bioavailability in rats was observed when HPMC was added to a conventional SEDDS formulation containing paclitaxel (Gao et al., 2003). The approach to combine LbDDS and PPIs was further investigated in multiple studies, all demonstrating the improved performance compared to PPI-free formulations, but still containing drug loads below the Seq in the preconcentrate (Gao et al., 2004; Suys et al., 2018). The current study aims at combining the advantages of super-SNEDDS and S-SEDDS, thereby enabling higher drug loads in the SNEDDS during storage and prolonging the supersaturated state

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after emulsification and in vitro lipolysis. Therefore, a PPI was added to a super-SNEDDS model system containing the poorly water-soluble drug SIM that was previously developed by Thomas et al. (2013). Due to its high solubility in the preconcentrate and its ability to generate an isotropic and monophasic formulation after dissolution, the widely used PPI polyvinylpyrrolidone-co-vinyl acetate (PVP/VA 64 consisting of 60 % VP and 40 % VA) was utilized as PPI for the supersaturated drug concentrations in the anhydrous preconcentrate, as well as during in vitro lipolysis. The prepared PPI super-SNEDDS, containing different drug loads and PPI concentrations, were characterized with regard to physical stability, rheological properties, droplet size after emulsification and performance during dynamic in vitro lipolysis.

2. MATERIALS AND METHODS 2.1 Materials Simvastatin (SIM) was purchased from Hangzhou Dayangchem (Hangzhou Ciy, China). Capmul MCM (medium chain (MC) mixed glycerides) and Captex 300 (MC triglycerides) from Abitec (Columbus, OH, USA) were kindly provided by Barentz (Odense, Denmark). Kolliphor RH 40 (polyoxyl 40 hydrogenated castor oil) and Kollidon VA 64 (polyvinylpyrrolidone-co-vinyl acetate) were donated by BASF (Ludwigshafen, Germany). Bovine bile extract (B-3883), porcine pancreatic lipase extract (P-1625), maleic acid, tris base and 4-bromobenzeneboronic acid (4-BBBA) were purchased from Sigma–Aldrich (Saint Louis, MO, USA) and soy phospholipid (S-PC) was obtained from Lipoid (Ludwigshafen, Germany). Sodium hydroxide, calcium chloride dihydrate and sodium chloride were purchased from Merck (Darmstadt, Germany). Ethanol (HPLC grade), methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from VWR (Herlev, Denmark).

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2.2 Methods 2.2.1 Preparation of formulations The SNEDDS preconcentrate was prepared as previously described by Thomas et al. (2013). The MC mixed glycerides (Capmul MCM) and the surfactant Kolliphor RH40 were molten at 50 ˚C and blended in a vortex mixer with the MC triglycerides (Captex 300). After equilibration to room temperature the co-solvent EtOH was added to the mixture followed by a second mixing step forming an isotropic preconcentrate consisting of 55 % (w/w) lipid (Capmul MCM : Captex 300 ratio 2:1), 35% (w/w) surfactant (Kolliphor RH40) and 10% (w/w) co-solvent (EtOH). Drug- and PPI-loaded SNEDDS preconcentrates were produced using a heating-cooling cycle. The required amounts of SIM, PVP/VA 64 and blank preconcentrate were weighed into Teflon-sealed glass vials containing a magnetic stirring bar. The mixtures were stirred at room temperature for 5 min and subsequently ultrasonicated in a Branson 5510 ultrasonic bath (Branson Ultrasonics, Danbury, CT, USA) for another 5 min. To facilitate complete dissolution of drug and PPI, the obtained suspensions were placed in a silicon oil bath (60 ˚C) and stirred for 2 h. After the end of the heating cycle, the stirring bar was removed and the vials were purged with nitrogen to prevent lipid oxidation during storage. Next, the prepared formulations were allowed to slowly equilibrate to room temperature inside the oil bath resulting in an isotropic drug- and PPI-loaded super-SNEDDS preconcentrate. The complete dissolution of SIM after the heating-cooling cycle was assessed using polarized light microscopy. Formulations used for the physical stability assessment were produced in triplicates.

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2.2.2 Determination of equilibrium solubility The Seq of crystalline SIM in blank SNEDDS preconcentrate and SNEDDS preconcentrate containing PVP/VA 64 was quantified using a shake flask method adopted from Thomas et al. (2012). Test tubes containing 1 g of the SNEDDS preconcentrate and an excess amount of SIM were constantly shaken on a test tube rotator at 25 ˚C (Heto, Birkerød, DK) and samples were withdrawn in regular intervals. After centrifugation at 13,300 rpm (17,000 x g) for 20 min and appropriate dilution of the obtained supernatant with acetonitrile the samples were quantified by HPLC (see below). The Seq of SIM was presumed to have been obtained when the concentration of two consecutive measurements did not vary by more than 5 %. The same method was employed to determine the Seq of SIM in fasted-state simulated intestinal fluid (FaSSIF) and in the digestion products of blank SNEDDS preconcentrate generated during dynamic in vitro lipolysis. All measurements were performed in triplicates.

2.2.3 HPLC analysis The samples obtained from the Seq experiments and during dynamic in vitro lipolysis (see below) were analyzed using an Agilent 1260 Infinity chromatographic system (Agilent Technologies, Santa Clara, CA, USA) equipped with an ACE C18 column (Advanced Chromatography Technologies Ltd, Aberdeen, Scotland). The used conditions were derived from a previously validated method (Kang et al., 2004). The mobile phase consisted of acetonitrile/water (80:20% V/V) at a flow rate of 1.2 ml/min and the eluted SIM was detected at a wavelength of 238 nm.

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2.2.4 Rheology The rheological properties of the produced formulations were assessed using a Discovery HR-3 rheometer (TA instruments, New Castle, DE, USA) equipped with a 40 mm cone plate geometry (cone angle of 1.002˚). Approximately 300 µl of the formulation was added to the steel Peltier plate, which was maintained at 25˚C during the measurement. After trimming excess formulation, the head was lowered to the measurement gap of 25 µm and a logarithmic flow sweep from a shear rate of 1 to 1000 s-1 was performed. All measurements were performed in triplicates.

2.2.5 Stability assessment of super-SNEDDS and PPI super-SNEDDS The SIM loaded super-SNEDDS and PPI super-SNEDDS were stored in sealed glass vials at 25 ˚C for up to 6 months to assess their physical stability. The vials were analyzed in regular intervals for possible precipitation of the dissolved drug by both visual observation and polarized light microscopy (see below). To ensure the chemical stability of the lactone ring in SIM after the employed heating cycle the content of SIM was quantified using HPLC directly after the equilibration to room temperature.

2.2.6 Droplet size measurements The droplet size after dispersion of the formulations was measured by dynamic light scattering (DLS). A USP type II apparatus (Erweka DT600 dissolution tester, Erweka GmbH, Heusenstamm, Germany) was used to promote emulsification in Milli-Q water. The amount of formulation used for the dispersion experiments corresponded to the ratio of formulation to aqueous media also used

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during dynamic in vitro lipolysis (see below). The appropriate amount of formulation was weighed into the dissolution vessel and 150 ml of preheated Milli-Q water was added. The emulsions were stirred at 100 rpm for 15 min at 37 ˚C and immediately analyzed using a Zetasizer Nano (Malvern, Worcestershire, UK) operated at 37˚C. The measured droplet size and polydispersity of three independent measurements per formulation is reported as mean z-average value and polydispersity index (PdI), respectively.

2.2.7 Dynamic in vitro lipolysis Dynamic in vitro lipolysis was carried out as described by Zangenberg et al. (2001) with minor modifications. The formulations were added to a temperature-controlled glass vessel (37 ˚C) resulting in the same amount of SIM and preconcentrate present during every lipolysis run for formulations of the same drug load. A dispersion step was employed to facilitate complete dispersion of the formulations before the start of digestion. At time 0 min, 25 ml of FaSSIF (bile salt 2.95 mM, phospholipids 0.26 mM, sodium chloride 50 mM, tris-maleate buffer 2 mM pH 6.5) were added to the lipolysis vessel equipped with an electrical stirrer. After 15 min of dispersion, 5 ml of freshly prepared pancreatic extract (600 USP/ml) were added to the intestinal medium to initiate lipid digestion. The rate of lipid digestion was controlled by the continuous addition of calcium (0.5 M) at a rate of 0.01 ml/min. A pH-stat apparatus (Metrohm Titrino 744, Tiamo Version 1.3, Herisau, Switzerland) was used to maintain a pH of 6.5 during the total run time of the experiment, dosing 0.4 M NaOH solution to compensate for the drop in pH caused by the release of free fatty acids. Samples were withdrawn after 5, 10 and 15 min of dispersion and after 5, 15, 30, 45 and 60 min of digestion. The lipase activity in the collected samples was directly inhibited by

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the addition of 5 µl 4-BBBA (1 M, dissolved in MeOH) and centrifuged at 17,000 g for 15 min, resulting in the formation of a pellet and clear supernatant. The solubilized amount of SIM in the supernatant was quantified using HPLC, after appropriate dilution with acetonitrile (see above). After 60 min of digestion, 15 ml of the lipolysis media was withdrawn from the vessel, added to 100 µl of 4-BBBA and centrifuged for 20 min at 5580 g. The isolated pellet was analysed by XRPD within 1 h after the end of digestion (see below). XRPD analysis was carried out for pellets obtained after the digestion of blank preconcentrate and drug- and PPI-loaded formulations.

2.2.8 X-ray powder diffraction The solid state of the isolated pellets generated after 60 min of dynamic in vitro lipolysis was analyzed by X-ray powder diffraction (XRPD). XRPD measurements were performed using a PANalytical X’Pert PRO X-ray diffractometer (PANalytical, The Netherlands) with Cu Kα radiation (λ = 1.542 Å, current 40 mA, voltage 45 kV), operated in reflection mode. The isolated pellets were placed on aluminum sample holders and scanned from 5 to 35 ° (2θ) using a scan speed 0.067335 ° s-1 and step size of 0.0262606 ° (2θ). The diffractograms of pellets obtained from drugand PPI-loaded formulations were analyzed within 1 h after lipolysis and compared with drug-free and crystalline SIM spiked pellets to assess the physical state of the precipitate.

2.2.9 Polarized light microscopy Drug-loaded formulations were analyzed for undissolved crystalline SIM after production and for crystalline precipitate upon storage using a Leica DM LM microscope equipped with cross polarizers (Leica Microsystems, Wetzlar, Germany). Images were acquired using a Media

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Cybernetics Evolution MP digital camera and the Image-Pro Insight software version 8.0 (Media Cybernetics).

2.2.10 Statistical analysis GraphPad Prism (version 8.1.1, GraphPad software, San Diego, CA, USA) was used to analyze statistical differences between more than two groups using a one-way ANOVA (p = 0.05) including Tukey´s test.

3. RESULTS AND DISCUSSION 3.1. Solubility of SIM Table 1 summarizes the measured Seq of SIM in PPI-free and PPI super-SNEDDS preconcentrate. The determined Seq of SIM in PPI-free preconcentrate is in good agreement with the previously reported value at 25˚C (101.9 ± 2.3 mg/g) by Thomas et al. (2013). When increasing amounts of PVP/VA 64 were added, the Seq of SIM increased in comparison to the PPI-free preconcentrate, indicating a capability of the added PPI to increase the solubility of the drug in the preconcentrate. The increased Seq of SIM in the PPI super-SNEDDS preconcentrate resulted in a lowered effective degree of supersaturation, since all reported drug loads in this study refer to the Seq of SIM in PPIfree preconcentrate. In contrast to the high solubility in the lipophilic SNEDDS preconcentrate, the Seq of SIM in FaSSIF at 37˚C was determined at 10.7 ± 1.1 µg/g demonstrating the low solubility of SIM in aqueous media.

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Table 1. Equilibrium solubilities of SIM in SNEDDS preconcentrates at 25 ˚C. Solubilities are presented as mean ± SD (n=3). Medium SNEDDS preconcentrate

PVP/VA 64 content (% (w/w)) 0 1 10 20

Solubility (mg/g) 99.9 ± 2.2 99.5 ± 0.8 114.3 ± 1.1 126.3 ± 3.0

3.2 Viscosity of formulations The dissolved PVP/VA 64 in the PPI super-SNEDDS increased the Seq of SIM in the SNEDDS preconcentrate and thus lowers the thermodynamic driving force for recrystallization of the supersaturated drug. Additionally, it can be assumed that the increase in viscosity due to the dissolution of large quantities of PPI in the preconcentrate (up to 20 % (w/w)) also plays a role in preventing the precipitation of the drug from highly supersaturated concentrations by lowering the molecular mobility and resulting in a kinetic stabilization of the formulations. The effect of different drug loads and PPI concentrations on the viscosity of the SNEDDS preconcentrate at 25˚C is shown in Figure 1. By only dissolving SIM in the SNEDDS preconcentrate (drug load 200 %, 0 % (w/w) PVP/VA 64), the viscosity increased more than twofold compared to the blank SNEDDS preconcentrate. The same thickening effect of the dissolved drug was observed when comparing the viscosities of PPI super-SNEDDS containing the same amount of PPI, but different drug loads of SIM. Despite the tendency of PPI super-SNEDDS containing 20 % (w/w) PVP/VA 64 to show shear thinning-behavior at high shear rates (> 700 s-1), the viscosities of all formulations remained constant over the entire range of shear rate, thus showing Newtonian flow behavior (see supplementary information, Figure S1). In course of the viscosity increase due to the

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large quantities of dissolved PPI and drug in the SNEDDS preconcentrate, concerns regarding the processability, e.g. capsule filling, may arise. However, studies conducted by Walters et al. (1992) demonstrate the possibility of filling hard gelatin capsules with Newtonian liquids in the viscosity range up to 25 Pa*s, which is well above the highest measured viscosity in this study of approx. 5 Pa*s.

Figure 1. Viscosity of blank SNEDDS preconcentrate and PPI- and drug-loaded SNEDDS preconcentrates at a shear rate of 100 s-1 measured at a constant temperature of 25 ˚C. The data is grouped according to SIM drug load and PVP/VA 64 concentration in the SNEDDS preconcentrates, with increasing PPI concentration from left (white column, 0 % (w/w)) to right (dark grey column, 20 % (w/w)). PPI-free super-SNEDDS with a drug load of 250 % were not included in the measurements due to a fast drug recrystallization. Results are presented as mean ± SD (n=3). 3.3 Stability of formulations

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Due to the high degree of supersaturation of the drug in the preconcentrate, the physical stability of the prepared formulations is of significant interest. Table 2 provides an overview of the investigated formulations and the corresponding onset of precipitation. The onset of precipitation in a formulation was defined as the time point when crystals were detected by PLM in at least one of the prepared triplicates. Within less than 24 hours, needle-shaped crystals were observed in PPIfree super-SNEDDS accompanied by a rapid recrystallization of the supersaturated drug fraction (Figure 2 a). Formulations with a drug load of 250 % were less physically stable compared to formulations with a drug load of 200 %, demonstrating the increased thermodynamic driving force towards recrystallization of SIM due to the higher degree of supersaturation. However, by dissolving 20 % (w/w) PVP/VA 64 in the preconcentrate, the physical stability of the PPI superSNEDDS was maintained for up to 6 months, whereas PPI super-SNEDDS with a drug load of 250 % showed the same tendency for faster precipitation, as seen for the PPI-free equivalents. Not only the onset of precipitation was delayed due to the additional dissolution of PVP/VA 64 to the SNEDDS, but also the macroscopic appearance and growth kinetics of the precipitate changed. As seen in Figure 2 b, spherical crystals that slowly grew outwards from a central point were observed for the PPI containing SNEDDS. Additionally, the rate of crystallization appeard to be slower compared to the PPI-free formulations (visual observation). These observations can be explained by the increased viscosity of the PPI super-SNEDDS (see section 3.2) leading to a reduced diffusivity of the drug molecules and thereby slowing down crystal growth, but also by the known ability of PPIs like PVP/VA 64 to adsorb onto the crystal surface and thereby altering the crystal habit (Brouwers et al., 2009).

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Directly after the production of the formulations, an average of 94.2 ± 1.8 % of the claimed SIM content was found in the super-SNEDDS and both PPI super-SNEDDS, indicating a reasonable chemical stability of SIM after the employed heating-cooling cycle.

Table 2. Physical stability of super-SNEDDS and PPI superSNEDDS stored at 25 ˚C (n=3). Drug load 200 %

250 %

PVP/VA 64 content (% (w/w)) 0 1 10 20 0 1 10 20

Onset of precipitation (days) 0.5 5 > 180 > 180 0.1 1 19 > 180

a)

b)

0.25 mm

0.25 mm 0.25 mm

Figure 2: Representative PLM micrographs of precipitate observed in PPI-free super-SNEDDS preconcentrates a) super-SNEDDS (200 % drug load) and PPI-loaded super-SNEDDS preconcentrate b) PPI super-SNEDDS (200 % drug load, 1 % (w/w) PVP/VA 64), both after 5 days of storage. The respective crystal habits were not affected by drug load or PPI concentration.

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3.4. Droplet size after dispersion The influence of drug load and PVP/VA 64 concentration on the droplet size after emulsification of the formulations in Milli-Q water is shown in Figure 3. Previous studies analyzing the same type of MC SNEDDS preconcentrate containing SIM at drug loads ranging from 25 % to 100 % found only minor changes in droplet size when the drug load of SIM in the formulation was increased(Thomas et al., 2012b). The results of the current study clearly demonstrate that drug loads above the Seq of SIM in the SNEDDS preconcentrate result in a significantly increased droplet size after emulsification (p < 0.05). In contrast, varying PPI concentrations had no significant influence on the droplet size of formulations with a drug load of 200 % (p = 0.58) and only caused a negligible size difference for drug-free and 250 % drug-loaded formulations. This finding indicates that the PPI is not incorporated into the emulsion droplets and most likely immediately dissolves into the aqueous phase after exposure to aqueous media. The time needed for a complete emulsification of the formulations varied depending on the concentration of PVP/VA 64. A complete emulsification was assumed if no SNEDDS preconcentrate was observed on the bottom of the dissolution vessel. Blank SNEDDS preconcentrate and PPI-free super-SNEDDS (200 % drug load) emulsified completely in less than 5 min. For the highly viscous formulations containing 20 % (w/w) PVP/VA 64, the time until complete emulsification was extended to more than 10 min. No measurable influence of the drug load on the emulsification time was observed. The generated nanoemulsion showed a clear blueish appearance for blank SNEDDS preconcentrate and a still transparent but white-blueish appearance for super-SNEDDS and PPI super-SNEDDS. Due to the large amounts of SIM dissolved in the PPI super-SNEDDS and the dilution of the cosolvent ethanol, recrystallization of the drug during the dispersion study was a major concern.

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However, no SIM crystals were detected by PLM after 15 min of stirring in the USP-II apparatus, enabling further analysis of the emulsions using DLS. When extending the emulsification time to more than 30 min, fluffy white agglomerates were formed consisting of fine, needle-shaped SIM crystals showing a similar habit to those observed in PPI-free super-SNEDDS (see Figure 2 a).

Figure 3. Droplet size measured after dispersion of the formulations in Milli-Q water. The data is grouped according to SIM drug load and PVP/VA 64 concentration in the SNEDDS preconcentrates, with increasing PPI concentration from left (white column, 0 % (w/w)) to right (dark grey column, 20 % (w/w)). All investigated formulations formed monodisperse (PDI < 0.1) emulsions (data not shown). No measurements were performed for PPI-free super-SNEDDS with a SIM drug load of 250 % due to fast recrystallization after production. Results are presented as mean ± SD (n=3). 3.5. Solubilization of SIM during dynamic in vitro lipolysis The concentration of SIM in the aqueous phase during the dynamic in vitro lipolysis of superSNEDDS and PPI super-SNEDDS is shown in Figure 4. Due to the viscosity differences and

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consequently different times needed for a complete emulsification of the formulations (see section 3.2), a dispersion step was employed to generate comparable concentrations of SIM in the aqueous phase before initiating lipid digestion. During the dispersion of the formulations, both the superSNEDDS and PPI super-SNEDDS reached supersaturated concentrations in the lipolysis medium compared to the Seq of SIM in the lipolysis medium during digestion of a blank SNEDDS preconcentrate (black asterisk). The PVP/VA 64 in the PPI super-SNEDDS had no impact on the aDS during the dispersion step (Figure 4 a, aDS ~ 1.7). The higher aDS of PPI super-SNEDDS with a drug load of 250 % (Figure 4 b, aDS ~ 2.1) within the first 15 min can be explained by its ability to incorporate more drug in the same amount of formulation due the higher drug load. After the addition of 5 ml of pancreatic extract to the lipolysis vessel, the digestion of the emulsified lipids was started. The high lipase affinity to MC lipids and the large surface area created by the formation of nano-sized emulsion droplets is known to lead to rapid hydrolysis of triglycerides within the first minutes of the experiment (Christensen et al., 2004; Deckelbaum et al., 1990). This effect is also reflected by the large decrease in solubilization capacity for SIM in the digestion products of blank SNEDDS preconcentrate after the onset of digestion (Anby et al., 2012). The solubilization capacity of PPI-free super-SNEDDS (Figure 4 a, circle and solid line) decreased rapidly after the initiation of lipid digestion at 0 min, but was able to maintain its supersaturated concentration until 30 min of digestion. The same trend towards precipitation was observed for the three 200 % drug-loaded PPI super-SNEDDS (Figure 4 a, triangles), but to a much smaller extend, demonstrating the precipitation inhibitory effect of the PVP/VA 64 dissolved in the formulations. This effect was most pronounced for PPI super-SNEDDS containing 20 % (w/w) of PVP/VA 64 (Figure 4 a, triangle and solid line) maintaining a 2.5-fold higher degree of supersaturation (aDS ~ 4.3) after 15 min of digestion compared to the PPI-free formulation (aDS ~ 1.7). PPI super-

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SNEDDS containing 1 % and 10 % (w/w) PVP/VA 64 (Figure 4 a, triangle, dotted and dashed lines) also showed an improved performance over the PPI-free equivalent, but reached similar concentration levels of SIM after 30 min. Increasing the drug load of the formulations from 200 % to 250 % had no beneficial effect on the solubilization of SIM during the first 30 min of lipolysis. In contrast, the higher initial degree of supersaturation during the dispersion step potentially promoted the precipitation of the drug after the initiation of lipid digestion, due to an increased thermodynamic driving force towards the equilibrium. This might also explain why formulations with a drug load of 250 % and a PPI concentration of 1 % and 10 % (w/w) PVP/VA 64 (Figure 4 b, square, dotted and dashed lines) exhibited lower concentrations of SIM during the first 30 min of digestion compared to 200 % drugloaded formulations containing the same amount of PPI. As similar trend was observed for 250 % drug-loaded PPI super-SNEDDS containing 20 % (w/w) PVP/VA 64 (Figure 4 b, square and solid line). The degrees of supersaturation at 5 min (aDS ~ 3.9) and 15 min (aDS ~ 3.8) were lower than the ones observed for the 200 % drug-loaded formulation containing 20 % (w/w) PVP/VA 64.

Figure 4. Solubilization of SIM during disperison and digestion of super-SNEDDS and PPI super-SNEDDS. Formulations consisting of a) superSNEDDS with a drug load of 200 % (circle, solid line) and PPI super-SNEDDS (200 %, triangle)

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and b) PPI super-SNEDDS (250 %, square) containing 1 % (w/w) (dotted line), 10 % (w/w) (dashed line) and 20 % (w/w) (solid line) of PVP/VA 64 were analyzed. Super-SNEDDS with a drug load of 200 % (circle, solid line) is shown again for comparison in 4b. The grey shaded area represents the duration of the dispersion step, which was followed by 45 min of lipid digestion after the addition of the pancreatic extract at 0 min. The amount of formulation added to the lipolysis vessel for each run contained 16 mg SIM for drug loads of 200 % and 20 mg SIM for drug loads of 250 %, whereas the ratio between drug and PPI-free fraction of the preconcentrate was kept constant for formulations of the same drug load. The Seq of SIM at 37 ˚C in the lipolysis medium during digestion of a blank SNEDDS is represented by the black asterisk. Results are presented as mean ± SD (n=3). 3.6. Characterization of pellet obtained after dynamic in vitro lipolysis Figure 5 shows the diffractograms of the isolated pellets obtained after dynamic in vitro lipolysis. All investigated pellets showed characteristic SIM diffractions (compare with SIM spiked pellet) indicating the presence of crystalline SIM in the precipitate. This finding is not in agreement with the previously reported, fully amorphous precipitate obtained after dynamic in vitro lipolysis of MC SIM super-SNEDDS (200 % drug load) (Thomas et al., 2013). When comparing diffraction intensities, differences between the SIM loaded formulation and SIM spiked pellet were observed, indicating the formation of a partially crystalline pellet. The solid-state properties of the precipitate formed during lipid digestion are known to be dependent on the isolation procedure of the pellet phase. Especially a long preparation time can favor crystallization from a potential amorphous precipitate and therefore not adequately reflect the precipitation occurred during lipolysis (Khan et al., 2016). However, during our study special attention was payed to minimize the time needed for the pellet isolation procedure to preserve the solid-state properties of the precipitate and reduce the chance of recrystallization. We therefore assume that the solid state of the precipitate formed during the lipid digestion is also governed by the lipolytic conditions and the kinetics of lipid digestion, whereby a rapid onset and fast initial decrease in solubilization capacity for the drug can lead to an at least partially crystalline precipitate.

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Figure 5. XRPD patterns of pellets obtained after 60 min of intestinal dynamic in vitro lipolysis of blank preconcentrate, super-SNEDDS and PPI super-SNEDDS. The PPI content is displayed as weight percentage (% (w/w)) and the abbreviation “Dl” represents the SIM drug load of the investigated formulations. The shown diffractograms are representative for replicate XRPD runs of independent samples (n=2). CONCLUSIONS The current study demonstrates the ability of PVP/VA 64 to act as a precipitation inhibitor in SNEDDS preconcentrates, to stabilize PPI super-SNEDDS containing SIM at a drug load of 250% for at least 6 months, and to enable supersaturated drug loads that lead to a nearly instant recrystallization in the absence of the PPI. Further characterization of the prepared PPI superSNEDDS suggested a multimechanistic stabilization effect of the dissolved PVP/VA 64 including

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a reduced molecular mobility due to an increased viscosity of the SNEDDS preconcentrate and a higher Seq of the drug in the presence of the PPI, where the higher Seq can be attributed to PPI-drug interactions. An understanding of the nature of the PPI-drug interaction resulting in the increased Seq and the inhibition of nucleation and crystal growth requires further investigation. After the dispersion of the SNEDDS in aqueous medium, the droplet size was not affected by an increasing PPI content, but grew slightly with increasing drug load in the PPI super-SNEDDS. Moreover, higher PPI concentrations in the PPI super-SNEDDS had no impact on the initial solubilization of SIM in the aqueous lipolysis medium, but could maintain a 2.5-fold higher degree of supersaturation (20 % (w/w) PVP/VA 64 and 200 % drug load) during the first 15 min of lipid digestion in a dynamic in vitro lipolysis experiment, compared to the PPI-free equivalent formulation. A further increase of the drug load to 250 % resulted in a similarly fast precipitation of SIM during the first 30 min of lipid digestion, suggesting that a higher drug load not necessarily improves the in vitro performance of PPI super-SNEDDS. Nevertheless, the incorporation of PPIs in super-SNEDDS is a promising approach to enable high drug loads in the lipid formulation and stabilize supersaturated concentrations in vitro.

ACKNOWLEDGEMENTS The Lundbeck Foundation is acknowledged for financial support. Grant number: R218-2016-1252

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Graphical Abstract

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The authors declare no conflicts of interest.

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CRediT author statement Jacob Bannow: Conceptualization, Investigation, Formal analysis, Visualization, WritingOriginal draft preparation, Reviewing and Editing Yasin Yorulmaz.: Investigation, Formal analysis Korbinian Löbmann: Supervision, Reviewing and Editing Anette Müllertz: Conceptualization, Supervision, Validation, Reviewing and Editing, Methodology: Thomas Rades: Conceptualization, Validation, Writing- Reviewing and Editing, Supervision, Methodology, Project administration

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