The influence of crushing amorphous solid dispersion dosage forms on the in-vitro dissolution kinetics

Journal Pre-proofs The influence of crushing amorphous solid dispersion dosage forms on the invitro dissolution kinetics Timothy Pas, Selam Verbert, Bernard Appeltans, Guy Van den Mooter PII: DOI: Reference:

S0378-5173(19)30929-9 https://doi.org/10.1016/j.ijpharm.2019.118884 IJP 118884

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International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

2 October 2019 12 November 2019 13 November 2019

Please cite this article as: T. Pas, S. Verbert, B. Appeltans, G. Van den Mooter, The influence of crushing amorphous solid dispersion dosage forms on the in-vitro dissolution kinetics, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118884

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The influence of crushing amorphous solid dispersion dosage forms on the in-vitro dissolution kinetics Timothy Pas, Selam Verbert, Bernard Appeltans, and Guy Van den Mooter* Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Campus Gasthuisberg ON2, Herestraat 49 b921, 3000 Leuven, Belgium

Corresponding author: Guy Van den Mooter; Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Campus Gasthuisberg ON2, Herestraat 49 b921, 3000 Leuven, Belgium [email protected]; Tel.: +32 16 330304

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Abstract Solid dosage forms of amorphous solid dispersions (ASDs) have rarely been assessed for their crushability, although it might possibly be a more frequent practice than thought to facilitate oral administration in several clinical conditions (e.g. dysphagia) when no oral liquids of the same drug are available. Nevertheless, there are concerns that contraindicate these formulations’ modification by grinding. For example, amorphous-amorphous phase separation, induction of crystallization, decreasing particle sizes, etc. might occur during grinding without knowing the implications on bioavailability. Hence, in this study, Sporanox® (itraconazole), Intelence® (etravirine), Noxafil® (posaconazole) and Norvir® (ritonavir), were selected as “model” enabling formulations (based on ASD) to evaluate if this concern was justified. Their assessment in simple and biorelevant media by two-stage in-vitro drug-release testing was performed which resulted in strong suspicion that pulverization is contradicted for some of these formulations. Despite differences were observed, uncertainty remains on the clinical relevance of these data as by golden standard it should still be confirmed by bioequivalence trials. Keywords: Amorphous solid dispersions, enabling formulations, crushing, grinding, dysphagia, facilitated administration, …

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Chemical compounds with PubChem CID reference: Chemical compound Etravirine Itraconazole

PubChem CID 193962 3793

Chemical compound Ritonavir Posaconazole

PubChem CID 392622 468595

Non-common abbreviations: Abbreviations common in the field: A – Specific surface area of the drug particle ACN – Acetonitrile ASDs – Amorphous solid dispersions AUC – Area Under the Curve BCS – Biopharmaceutics classification system Cmax – Maximum concentration Cs – Thermodynamic solubility Ct – Drug concentration at time t D – Diffusion coefficient dM/dt - Dissolution rate e.g. – Exempli gratiā / for example F2-factor – Similarity factor FaSSGF – Fasted State Simulated Gastric Fluid FaSSIF – Fasted State Simulated Intestinal Fluid FDA – Food and Drug Administration FeSSIF – Fed State Simulated Intestinal Fluid GI-tract – Gastro-intestinal tract h – Diffusion layer thickness (or “hour”) HCl – Hydrochloric acid HIV – Human Immunodeficiency Virus HPC – Hydroxypropyl cellulose HPMC – Hydroxypropyl methylcellulose MCC – Microcrystalline cellulose NCEs – New chemical entities ND – Non-detected PS 80 – Polysorbate 80 PVPVA – Polyvinylpyrrolydon vinylacetate QC – Quality control RPM – Rounds per minute (RP)-HPLC – (reversed Phase) - High Performance Liquid Chromatography SGF – Simulated gastric fluid Tg – Glass transition tmax – Time at which max concentration is observed USP – United States Pharmacopeia UV/VIS – Ultraviolet/visible light V – Volume V/V% - Volume/volume percentage XRPD – X-ray (powder) diffraction γ – Surface (/interfacial) tension

Abbreviation uncommon in the field: -

1. Introduction

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Given its non-invasiveness, ease of administration, high patient acceptability, cost-effectiveness, and flexibility of dosage form design; oral drug delivery prevails as the most attractive route of administration (Aulton, 2008; Baghel et al., 2016)(Sastry et al., 2000). Despite these advantages, modern drug selection procedures progressively resulted in highly unfavourable physicochemical properties of new chemical entities (NCEs) limiting their gastro-intestinal solubility and/or permeability and hence restricted oral bioavailability (Lipinski, 2002; Thayer and Houston, 2010). As a matter of fact, ca. 40% of marketed and ≥ 60-70% of candidate drugs currently suffer from limited aqueous solubility (Biopharmaceutics Classification System (BCS) Class II (or IV)) (Benet, 2013; Thayer and Houston, 2010). In order to “enable” their oral administration, formulation scientists faced this challenge by creating innovative formulation technologies of which amorphous solid dispersions (ASDs) are very promising (Janssens and Van den Mooter, 2009; Leuner and Dressman, 2000; Van den Mooter, 2012; Zhang et al., 2018). ASDs are made up of a drug that is molecularly dispersed in a pharmacologically inert carrier, forming a glass solution. Compared to the poorly soluble crystalline materials, ASDs exhibit higher drug dissolution rates and solubilities due to increased free energy and as a consequence lead to increased oral bioavailability. In some clinical settings (geriatric and pediatric patients suffering from dysphagia and hospitalized patients that undergo nasogastric feeding, gastrostomy feeding or enteral intubation) oral solid dosage forms (tablets, pellets) are often being manipulated (e.g. crushed, opening of capsules, …), in absence of liquid oral or injectable formulations, to facilitate and ensure intake (Gill et al., 2011; Han Sol et al., 2019; Mercovich et al., 2014; Paparella et al., 2010; Salmon et al., 2013). In most of these situations, the modification of oral solid dosage forms is being regarded as “off-label use” which is not covered by a product’s original license (unless included in the package leaflet) making the prescriber and/or person undertaking the modification accountable (= legal liability) (Gill et al., 2011). Hence, altering solid oral dosage forms is not without consequences and many pitfalls have already been identified.

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The only way to justify oral solid dosage forms to be modified is for that reason through bioequivalence studies between the altered and unaltered drug product(s) or by using predefined sprinkle medication for which sprinkling is registered (Han Sol et al., 2019). To the best of our knowledge, oral solid dosage forms of ASDs have been neglected and/or underestimated so far in this discussion which is possibly due to illiteracy of health care providers on these “novel” systems. Nevertheless, from a physicochemical point of view, there are substantial concerns that contraindicate their modification (see Fig. 1). Particle size reduction upon crushing is a first one as it will impact the dissolution rate of a drug. In case of ASDs, this implies a faster increase in drug-release to reach supersaturation and hence more chance on early/quick nucleation and crystal growth potentially hampering their bioavailability (Baghel et al., 2018). Another concern, related to ASDs, is the induction of alterations to an amorphous drug’s solid-state by crushing as the application of mechanical stress is known to possibly induce crystallization of the drug and/or amorphous phase separation (Davis and Walker, 2018; Newman, 2015). These significant changes in a formulation’s physicochemical properties may also result in variable performance and hence hampered bioavailability. Furthermore, crushed ASDs might also suffer from impaired stability prior to administration depending on how they are handled. If too much time passes by between crushing and administration, crystallization or phase separation might be observed through plasticization effects of attracted water (Tg↓) originating from ASDs’ hygroscopic nature. Alternatively, any prolonged exposure within the liquid vehicle, intended for administration, is contraindicated and the occurrence of crystallization will only be a matter of time (time that is normally spend in the GI-tract). According to these points of consideration and the currently existing knowledge gap on the crushability of solid dosage forms of ASDs, major regulatory, efficiency and safety concerns are in need to be addressed! The objective of the present study was to investigate the influence of crushing of solid dosage forms of ASDs (tablets and capsules) on their in-vitro drug dissolution profile. Sporanox® (itraconazole, antifungal), Intelence® (etravirine, antiviral – anti-HIV), Noxafil® (posaconazole,

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antifungal), and Norvir® (ritonavir, antiviral – anti-HIV) were selected as model solid dosage formulations for in-vitro drug-release evaluation. Their assessment for equivalence after crushing was performed in both quality control (non-biorelevant) and biorelevant dissolution set-ups. 2. Materials and Methods 2.1. Materials Ritonavir and posaconazole were purchased from Sigma-Aldrich (Gillingham, UK). Itraconazole and etravirine were obtained from Janssen Pharmaceutica NV (Beerse, Belgium). Naproxen was supplied by Fagron NV (Waregem, Belgium). Their chemical structures can be found in Fig. 2. Dimethyl sulfoxide was purchased from Acros Organics (Geel, Belgium). Acetonitrile (ACN) was purchased from Fisher Scientific (Leicestershire, UK). Citric acid monohydrate and sodium chloride (NaCl) were purchased from Chemlab NV (Zedelgem, Belgium). Sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium acetate anhydrous and trisodium phosphate dodecahydrate (Na3PO4.12H2O) were acquired from Sigma-Aldrich (Steinheim, Germany and/or UK). Acetic acid was supplied by VWR (Leuven, Belgium). Sodium hydroxide pellets were purchased from BDH laboratory supplies (Poole, UK). Polysorbate 80 (PS 80) was purchased from Fagron NV (Nazareth, Belgium) and 1 M stabilized hydrochloric acid (HCl) from Fisher Scientific (Loughborough, UK). FaSSIF/FaSSGF/FeSSIF biorelevant powder was bought from Biorelevant.com Ltd (Croydon, UK). Hypromellose (= hydroxypropyl methylcellulose = HPMC 2910 – 5 mPa.s), lactose monohydrate, titanium oxide, talc, calcium hydrogen phosphate and magnesium stearate were kindly donated by Janssen Pharmaceutica NV (Beerse, Belgium). Macrogol (polyethylene glycol) 20000 and sodium dodecyl sulfate were acquired from Merck (Darmstadt, Germany). Microcrystalline cellulose (MCC), colloidal anhydrous silica and hydroxypropyl cellulose (HPC) were purchased from Federa SA (Brussels, Belgium). Kollidon 30 (= Polyvinylpyrrolydon = PVP K-30) and Kollidon VA 64 (= Polyvinylpyrrolydon vinylacetate = PVPVA) were purchased from BASF® ChemTrade GmbH (Ludwigshafen, Germany). Macrogol 4000 was acquired from Fagron NV (Waregem, Belgium). Sucrose was acquired from Acros Page 6 of 29

Organics (New Jersey, USA). Polyvinyl acetate (PVA) was purchased from Sigma-Aldrich (Saint Louis, USA). Hypromellose acetate succinate (= hydroxypropyl methylcellulose acetate succinate = HPMC-AS) was purchased from Shin-Estu Chemicals (Tokyo, Japan). Purified water (pH 6.05 ± 0.035, R> 18 Ohm) was generated by Maxima system (Elga Ltd., High Wycombe Bucks). 2.2. Marketed formulations Sporanox® 100 mg capsules (Janssen-Cilag NV, Beerse, Belgium), Intelence® 200 mg tablets (JanssenCilag NV, Beerse, Belgium), Noxafil® 100 mg tablets (MSD Belgium bvba, Heist-op-den-Berg, Belgium), and Norvir® 100 mg tablets (AbbVie SA, Wavre, Belgium) were delivered by the University Hospitals Leuven (Belgium). The batch numbers were IBL9700, HLL5300, 8NOXA02003, 1091435, respectively. Their qualitative composition of active pharmaceutical ingredient and excipients is provided in Table 1. 2.3. Solubility testing Thermodynamic solubilities of etravirine, itraconazole, posaconazole and ritonavir were determined with the shake-flask method in simulated gastric fluid (SGF) pH 1.2, in 20 mM phosphate buffer pH 6.8, in FaSSGF and in a mixture of FaSSGF and FaSSIF (1:3 ratio, V/V%). In addition, thermodynamic solubilities were also evaluated in presence of the formulation’s water-soluble excipients to address their effect on drug-solubility. The selected composition(s) of these excipient-containing media can be found in appendix A, Table A1 (supplementary information). With respect to etravirine and itraconazole, excess amount of drug was added to glass test tubes containing 10 mL of desired medium whilst for posaconazole and ritonavir, approximately 1 mg or 0.5 mg of drug was added to microcentrifuge tubes filled with 1.5 mL of medium. In order to improve the microcentrifuge tubes’ hydrodynamics, glass beads were added. The equilibration time was set at 48 hours. Before analysis, the undissolved fraction of drug was first removed by means of filtration through a 0.2 µm (filtropur S 0.2 filter with PES membrane from Sarstedt Aktiengesellschaft & Co. (Nümbrecht, Germany)) or a 0.45 µm (CHROMAFIL® 0-45/15 MS PTFE filter from MACHERY-NAGEL GmbH & Co. KG (Düren, Germany)) Page 7 of 29

filter. Filtrated fractions were subsequently diluted with the mobile phase prior to HPLC analysis. All experiments were performed in triplicate. 2.4. Sample preparation for drug-release testing The selected formulations were manually ground using mortar and pestle to reflect the clinical setting. Although crushing devices exist, mortar and pestle were preferred in order to rule out any confounding possibly attributed to different devices. Since drug-release testing was performed in sixfold, all tablets were crushed separately before testing by using clean materials. Intact formulations were used as reference(s). In case of Sporanox, capsules were opened and the pellets were crushed in order to avoid the lag-time of capsule opening upon dissolution so that observed changes could only be attributed to the crushing of the beads. One whole dosage form was used in each dissolution vessel. 2.5. Two-stage in-vitro drug-release testing 2.5.1. Non-biorelevant (Ph. Eur.) set-up A two-stage Quality-Control (QC) based dissolution test was performed under non-sink conditions using 1 L vessels. Gastro-intestinal environmental conditions were mimicked, according to the protocol defined in the European Pharmacopoeia (European Pharmacopoeia 9.0, 2017), by first filling them with 900 mL of simulated gastric fluid (SGF) at pH 1.2 followed by the addition of 0.5 M Na3PO4-solution (60 - 85 mL) after 1 h until pH 6.8 was reached in order to reflect gastric emptying to the duodenum. Experiments were started after preheating the gastric dissolution medium to 37 °C. Media were continuously stirred at 75 rpm and sampling was performed after 5, 15, 30, 50, 60, 70, 90, 120, 140, 180, 240 and 300 min. 1.5 mL samples were collected at each time point and the same volume of fresh dissolution medium was immediately added to replace withdrawn quantities. Subsequently, samples were centrifuged at 20817 x g for 7 minutes using Microcentrifuge 5424 (VWR International, Dublin, Ireland) and 1 mL of the supernatant was collected for filtration through a PTFE filter with a pore size of 0.2 µm (Whatman Inc., Clifton, NJ, USA). Afterwards, 500 µl of the filtrate was appropriately diluted with mobile phase or ACN prior to HPLC analysis. All filters were, prior to in-vitro Page 8 of 29

drug-release testing, pre-treated with drug-saturated aqueous solutions and subsequently firmly washed in order to prevent drug-adsorption. All experiments were performed in six-fold. 2.5.2. Biorelevant set-up Regarding the biorelevant two-stage dissolution set-up, a similar protocol as described in Section 2.5.1. was applied with minor adjustments. In order to be biorelevant, the non-biorelevant dissolution media were replaced by FaSSGF pH 1.6 and FaSSIF pH 6.5, their volumes were downsized to 50 mL (FaSSGF) and 150 mL (FaSSIF) to simulate the gastro-intestinal environment, customized 250 mL vessels (and dimensionally scaled paddles) instead of 1 L ones were applied and the stirring rate was modified to have comparable hydrodynamics (50 rpm instead of 75 rpm). Details of other relevant parameters like sampling, temperature and sampling volume are summarized in Table 2. The hydrodynamics of the smaller (250 mL) dimensionally scaled vessels and paddles (conform USP) were scaled to those of 1 L (USP). For the current project, dissolution profiles obtained for the poorly soluble drug naproxen in 900 mL and 955 mL of dissolution medium (50 mM sodium acetate buffer at pH 4.75 and 20 mM phosphate buffers at pH 5.5 or 6.0 with, if necessary, polysorbate 80) were compared with those resulting from 50 mL and 200 mL, respectively. Similarity was based on f2-value calculations. Biorelevant media were prepared according to the standardized protocol described on biorelevant.com. FaSSGF was obtained by dissolving 0.060 g FaSSIF/FeSSIF/FaSSGF of biorelevant powder and 1.999 g of sodium chloride in 1 L purified water with subsequent pH-adjustment to pH 1.6 with 1 M HCl. Concentrated FaSSIF (1.33-fold FaSSIF), on the other hand, was prepared by dissolving 2.98 g FaSSIF/FeSSIF/FaSSGF of biorelevant powder, 8.23 g of NaCl, 0.56 g of NaOH, and 5.25 g of NaH2PO4.H2O in 1 L purified water with subsequent pH-adjustment to pH 7.4. After fresh preparation, these media were left untouched for at least 2 h before use (only necessary for FaSSIF) and were kept at ambient temperature to be used within 48 h following their preparation. 2.6. Concentration determination - HPLC analysis Page 9 of 29

Samples were investigated for drug content by RP-HPLC with UV-VIS detection. All analysis were performed using a Nucleodur C18 gravity column (4.6 mm x 150 mm, 5 µm) connected to a MerkHitachi LaChrom system (a D-7000 interface, a L-7420 ultraviolet-visible detector, a L-7200 auto sampler and a L-7100 pump) and the flow rate was fixed at 1 mL/min. Isocratic methods were developed for each compound and were validated for linearity (R2 ≥ 0.9995 for compound specific concentration range), precision and accuracy. A summary of all chromatographic parameters for each compound can be found in Appendix B (supplementary data). Analysis of the collected results was managed by using Merck LaChrom D-7000 system Manager Software (version 4.1). 2.7. X-ray powder diffraction (XRPD) XRPD measurements were collected of the intact drug product, after crushing, and during and after in-vitro drug-release testing in order to identify potential solid-state transformations. In order to distinct between API and excipients, diffractograms of all (semi)-crystalline excipients were also recorded. An X’pert PRO diffractometer (PANalytical, Almelo, the Netherlands) equipped with a Cu tube (λ Kα1 = 1.5418 Å), a generator set at 45 kV and 40 mA, an X’pert PRO goniometer and an X’Celerator detector was used. Prior to analysis, ground, intact or undissolved fractions of the selected solid dosage forms were placed between Kapton® polyimide films CAT. NO.: 446-5-P01 (PANalytical, USA) and were loaded in a sample holder. All experiments were conducted in transmission mode using a continuous scan mode from 4° to 40° 2θ with 0.0167° step size and 400 s counting time at room temperature. Data acquisition was subsequently performed using the X’pert Data viewer software (PANalytical, Almelo, The Netherlands). 2.8. Data analysis

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The hydrodynamic similarity between both types of dissolution vessels (dimensionally scaled) was evaluated by calculating similarity factors of dissolution profiles (f2-factors - Eq. 1) (Shah et al., 1997). ଵ ௡

ି଴Ǥହ

݂ଶ ൌ ͷͲ ൈ Ž‘‰ሾቀͳ ൅ ቀ ቁ σ௡௧ୀଵሺܴ௧ െ ܶ௧ ሻଶ ቁ

ൈ ͳͲͲሿ (Eq. 1)

Where n is the number of sampling time points, Rt is the concentration of dissolved drug (expressed in percentage) at each of the selected time points of the reference and Tt represent those of the test product, respectively. F2-factors between 50 and 100 can be regarded as similar, according to the Food and Drug Administration (FDA). An f2-value of 50 corresponds to not more than 10% of average difference at each time point (Shah et al., 1997). Furthermore, two-tailed student t-tests were applied to evaluate if significant differences in concentration existed at each time point of in-vitro drug-release testing between crushed and intact solid dosage forms. A p-value ≤ 0.05 was set as the level of significance.

3. Results and Discussion 3.1. Scaling of hydrodynamics In order to exclude hydrodynamics being a significant reason of observed differences in in-vitro drugrelease testing, naproxen was selected as poorly soluble BCS Class II drug to scale its dissolution behavior for both dissolution settings to result in similar hydrodynamics (based on f2–factors). Naproxen’s profile of dissolution was first tailored (by means of pH, surfactants, etc.) to gradually increase over the different time points to enable a meaningful evaluation since, depending on the conditions, poor wettability of naproxen hampered valid assessment of hydrodynamics. In the end, similarity in hydrodynamics (900 mL vs. 50 mL and 955 mL vs. 200 mL) was evaluated in 20 mM phosphate buffer pH 5.5 with 0.25 µg/mL PS 80. Similar hydrodynamics were obtained (f2-factors of 79.91 % and 92.78 % (Table 3)) for the small (250 mL) and conventional large (1 L) vessels when stirred at 50 and 75 RPM, respectively.

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3.2. Solubility The thermodynamic solubility data of the selected drugs in presence or absence of water-soluble excipients is summarized in Fig. 3 (Numerical values can be found in Appendix A, Table A2, Supplementary information). In case of addition of excipients, the amount of excipient added was chosen based on its aqueous solubility and its viscosity in solution. In order to assess the excipients’ effect on the drugs’ solubility, the excipients’ concentration was strived to be as high as feasible. The solubility of etravirine could not be detected in SGF, phosphate buffer pH 6.8 and FaSSGF. However, enhanced solubilities were observed in presence of HPMC. For the weakly basic drugs (itraconazole, posaconazole and ritonavir), on the other hand, higher solubilities in SGF and FaSSGF compared to neutral media were noticed as expected. For ritonavir, it was in particular observed that the addition of excipients did not significantly affect its solubility in neutral media (pH 6.80 and FaSSGF+FaSSIF) apart from a slight decrease in solubility in presence of 2.5% HPMC (and hence also the mixture). In case of posaconazole, the presence of Noxafil®’s water-soluble excipients mainly negatively impacted the solubility of posaconazole in SGF and FaSSGF+FaSSIF. 3.3. Two-stage in-vitro drug-release testing 3.3.1. Intelence® This ASD, comprising the weakly basic drug etravirine (pKa-values of 4.1 and 12.5) and HPMC as inert carrier, displayed opposite dissolution behavior in both set-ups (see Fig. 4). In the QC-based method, maximal supersaturation (± 0.10 % of the drug’s dose) was generated in SGF within the first 5-15 min followed by gradual precipitation, which further progressed towards the limit of quantification of etravirine after neutralization. As illustrated in Fig. 3, etravirine’s equilibrium solubility could not be detected in both dissolution media. Comparison of ground and intact tablets of Intelence® in this QCbased set-up resulted in a statistically significant difference for drug concentrations in SGF exceeding 15 min of dissolution. Overall, a minor increase in concentration was observed in case tablets were ground. Page 12 of 29

In contrast to the above QC-based in-vitro dissolution test, remarkably low drug-release of etravirine was attained in FaSSGF, whilst the maximum dissolved amount of etravirine in FaSSGF+FaSSIF approximated > 100 times that in FaSSGF (see Fig. 4). This phenomenon is likely to be attributed to the presence of more micelles in FaSSGF+FaSSIF compared to FaSSGF, enhancing the apparent solubility. There appeared to be no significant difference in in-vitro drug-release whether or not Intelence® was crushed. In both conditions, drug-release profiles were characterized by a decline in concentration towards the drug’s equilibrium solubility in both media after reaching their achievable upper limit of supersaturation (Fig. 4) (from ± 0.33 µg/mL towards ± 0.156 µg/mL in FaSSGF and from ± 30 µg/mL towards ± 11 µg/mL in FaSSGF+FaSSIF). Despite declines in concentration were observed, Intelence® (crushed or not) generated supersaturated etravirine levels over the whole timeframe of in-vitro drug release testing.

3.3.2. Norvir® The ASD-based film-coated tablets (Norvir®) containing the weakly basic drug (pKa 2.8 and 13.7) ritonavir, resulted in both set-ups (QC and biorelevant) in a more pronounced burst-release-like dissolution for ground tablets which was followed by elevated precipitation kinetics in the gastric compartment (see Fig. 5). Most likely the destruction of the film-coating upon crushing is (partially) responsible for this observation as by default this affects release kinetics of a drug from a formulation. Although no functional coating is concerned, its destruction has resulted in altered dissolution kinetics probably due to loss of swelling and dissolution of the coating itself and the lacking need of disintegration since a (crushed) powder instead of a tablet is concerned (difference in surface area and porosity). Hence, these observations are in accordance with the drug’s leaflet as it indicates not to chew, break or crush these tablets. Nevertheless, it is hard to predict of the same observation can be translated to in-vivo. In the duodenal compartment after transfer from the acidic compartment, no

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significant differences in obtained levels of drug-release were detected. Higher drug concentrations for Norvir® compared to QC-testing were attained in biorelevant dissolution media. 3.3.3. Noxafil® In-vitro drug-release evaluation of Noxafil® tablets containing molecularly dispersed posaconazole (pKa’s = 3.6 and 4.6) in a HPMC-AS matrix (pH-sensitive carrier that dissolves at pH > 5.5), showed significantly increased drug-release in SGF and FaSSGF when ground (see Fig. 6). However, supersaturation was not induced in this gastric compartment due to the pH-dependent solubility of HPMC-AS and posaconazole. Approximately a 30 % to 90 % increase in drug concentration due to grinding was observed in both acidic conditions, probably induced by the increased surface-area-tovolume ratio resulting in facilitated release. Compared to the biorelevant set-up, a gastric biphasic increase in in-vitro dissolution was observed instead of a monophasic one of which the reason remains to be investigated as it was observed for both intact and crushed Noxafil®. Subsequent simulation of gastric emptying with its associated neutralization, led to generation of supersaturation in the intestinal-mimicking media with normalization of posaconazole in solution to the same level that was followed by gradual precipitation towards the thermodynamic solubility of posaconazole in pH 6.80 and FaSSGF+FaSSIF. Interestingly, both solubility and dissolution of this drug in the biorelevant (fasted) set-up were significantly lower (> 2- to 3-fold) than in case of QC-testing. Accordingly, the leaflet advises administration in the fed-state to sufficiently increase oral uptake of the drug. Hence, these model in-vitro set-ups apparently were too simplistic for Noxafil®. 3.3.4. Sporanox® For Sporanox®, a bead-coated ASD of itraconazole (pKa = 2.0 and 3.7) and HPMC on sucrose beads, capsules were dismantled to be used in both conditions (intact vs. crushed) to prevent the occurrence of the lag-time of capsule opening to be of any significant impact. Supersaturation of itraconazole was generated and preserved in all conditions over the full length of the in-vitro dissolution experiments (see Fig. 7). In SGF, a maximum of 88% of the drug was released Page 14 of 29

within 1 h. However, ground beads displayed a more rigorous increase in dissolution to approach this maximum and significantly higher concentrations for the 5 min and 15 min time point were observed. After neutralizing to pH 6.80, concentrations dropped due to its pH-dependent solubility and started to approach itraconazole’s thermodynamic solubility in this medium. The same trend was observed when biorelevant settings were applied. However, in this case no significant difference between the intact and ground beads could be established. Although the addition of FaSSIF to FaSSGF also resulted in a drop in concentration, slightly increased drug-release was observed during this transition (4 times dilution, 50 mL → 200 mL upon neutralization). Nevertheless, sustained contact with this medium also led to moderate precipitation towards the equilibrium solubility.

3.4. Solid-state analysis using X-ray powder diffraction (XRPD) XRPD-analysis of intact and crushed formulations was implemented for solid-state characterization in order to investigate if the applied mechanical stresses during grinding compromised the amorphous state of the intact enabling formulations. With the exception of Norvir®, diffractograms did not show any change in Bragg peaks before and after crushing (See appendix C, supplementary information). In case of Norvir®, grinding resulted in suppressed presence of Bragg peaks of the opacifier titanium dioxide (most intense for 25.20, 36.78, 37.61 and 38.38 °2∂) as a result of its dilution with the tablet’s core. Overall, it was revealed that only excipient-based changes had occurred and that all tested ASDformulations conserved the amorphous state of their drug upon grinding (all Bragg peaks could be associated to presence of (semi)-crystalline excipients). In addition, collected precipitates during and after the in-vitro drug-release experiments were also characterized by means of XRPD to detect solid-state transformations during dissolution. In general,

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both intact and ground drug products exhibited similar solid-state behavior during dissolution. All dosage forms appeared X-ray amorphous, even upon crushing. 3.5. Clinical relevance and limitations The current in-vitro dissolution experiments demonstrated significantly affected drug-release kinetics of e.g. Intelence®, Noxafil®, Norvir® and Sporanox® by the act of crushing. However, only moderate differences were concerned as two-stage in-vitro dissolution testing is only a very simplistic representation of the complex intraluminal environment of the human GI-tract (Boyd et al., 2019). Nevertheless, these significant, but modest, changes in drug-release might be of greater clinical impact than originally deducted from these results as the second hurdle of bioavailability (“permeability”) is neglected in these set-ups. Nonetheless, the significance of simultaneously occurring permeation in this particular case remains to be investigated in-vitro. It is presumed that concurrent withdrawal of dissolved drug through permeation will result in creation of sink conditions which might unveil more considerable divergence to strengthen the observations made in this study. Though, the necessity of bioequivalence studies exists to be of real clinical relevance, such that drug-release kinetics, pharmacokinetics and bioavailability can be assessed between intact and crushed tablets. Therefore, clinical trials are being considered to be performed in the near future. To date, few clinical studies have been performed to address the effects of grinding of ASDs on the drug’s pharmacokinetics and directly correlated pharmacodynamics. The open-label, cross-over clinical study conducted within pediatric patients to compare the pharmacokinetics of intact and crushed Kaletra® (lopinavir/ritonavir) tablets showed decreased AUC (approximately 40% after crushing) for both lopinavir and ritonavir. Hence, in this particular case, crushing resulted in lower systemic exposure and hence higher risk for development of viral resistance and treatment failure (Best et al., 2011). Secondly, a phase I/II, open-label, pharmacokinetic study to treat HIV-infected children below the age of six, using Intelence® “dispersible” tablets, has also already been performed.

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In this study, comparable pharmacokinetics were observed between the intact and dispersed tablets as dispersible tablets are concerned. 4. Conclusion The collected in-vitro dissolution data clearly pointed out that significant, though modest differences could be observed between intact and crushed ASD-based formulations. In particular, Norvir® and Noxafil® showed distinctive dissolution behavior. In addition, dissimilarities between both dissolution set-ups were also recognized stressing the added value of biorelevant testing. When translating these observations into the in-vivo situation, there is concern that the drugs’ pharmacokinetics might potentially be negatively impacted by crushing through altered dissolution kinetics compared to the intact enabling formulations. Hence, based on these findings, it is urged that one has to be cautioned to crush any type of enabling formulation since the outcome might be unpredictable as well as because of legal liability concerns. Clinical studies addressing bioequivalence should be conducted for each formulation separately to exclude any uncertainty and provide a definitive formulation-specific answer. Such clinical trials are being considered to be performed in the near future. Figure captions: Figure 1: Amorphous solid dispersions based technologies: stability and impact on solubility. The basics of these formulations question these formulations’ crushability to facilitate oral drug administration. (Figure explaining the generalized mechanism of drug supersaturation generation and maintenance during the dissolution of amorphous drugs and ASDs was copied with permission from Pas et al. (Pas et al., 2019). Figure 2: Chemical structures of etravirine, intraconazole, posaconazole and ritonavir. Created with MarvinSuite software (ChemAxon).

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Figure 3: Equilibrium solubility data, plotted as histograms, of etravirine, itraconazole, posaconazole and ritonavir in different dissolution media with or without certain excipients (n=3). (Numerical values can be found in Appendix A, Table A2, Supplementary information) Figure 4: In-vitro dissolution profiles of Intelence® during QC set-up (left) and biorelevant set-up (right) (n=6, unless otherwise indicated). Legend: Black profiles = intact formulation, Red profiles = crushed formulation, blue profiles = limit of quantification (LOQ) or Limit of detection (LOD) of etravirine in HPLC-analysis and purple/pink profiles = solubility of etravirine in FaSSGF+FaSSIF. Figure 5: In-vitro dissolution profiles of Norvir® during QC set-up (left) and biorelevant set-up (right) (n=6, unless otherwise indicated). Legend: Black profiles = intact formulation, Red profiles = crushed formulation, blue profiles = solubility of ritonavir in SGF (left) or FaSSGF+FaSSIF (right) and purple/pink profiles = solubility of ritonavir in pH 6.8 (left) or FaSSGF+FaSSIF (right). Figure 6: In-vitro dissolution profiles of Noxafil® during QC set-up (left) and biorelevant set-up (right) (n=6, unless otherwise indicated). Legend: Black profiles = intact formulation, Red profiles = crushed formulation, blue profiles = solubility of posaconazole in SGF (left) or FaSSGF+FaSSIF (right) and purple/pink profiles = solubility of posaconazole in pH 6.8 (left) or FaSSGF+FaSSIF (right). Figure 7: In-vitro dissolution profiles of Sporanox® during QC set-up (left) and biorelevant set-up (right) (n=6, unless otherwise indicated). Legend: Black profiles = intact formulation, Red profiles = crushed formulation, blue profiles = solubility of itraconazole in SGF (left) or FaSSGF+FaSSIF (right) and purple/pink profiles = solubility of itraconazole in pH 6.8 (left) or FaSSGF+FaSSIF (right). Table captions: Table 1: Detailed information on Sporanox®, Intelence®, Noxafil® and Norvir®. The active pharmaceutical ingredient (drug), its physicochemical properties, a qualitative list of excipients, the dose strengths available, the dosage form and whether the formulation may be crushed according to the leaflet are provided.

Page 18 of 29

Table 2: Detailed overview of both two-stage in-vitro drug-release testing set-ups. Table 3: Summary of obtained final f2-factors based on in-vitro dissolution data. In their comparison by f2-factors, the 900 mL and the 955 mL vessels were considered as reference in the calculations, whilst the others as test conditions. Abbreviations: (S) = small vessels (250 mL vessel – 50 or 200 mL of dissolution medium), (C) = conventional large vessels (1 L vessel – 900 or 955 mL of dissolution medium).

References: Aulton, M., 2008. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines: 3rd (third) Edition, London: churchill livingstone elsevier. https://doi.org/10.1016/0168-3659(89)90050-3 Baghel, S., Cathcart, H., O’Reilly, N.J., 2018. Understanding the generation and maintenance of supersaturation during the dissolution of amorphous solid dispersions using modulated DSC and1H NMR. Int. J. Pharm. 536, 414–425. https://doi.org/10.1016/j.ijpharm.2017.11.056 Baghel, S., Cathcart, H., O’Reilly, N.J., 2016. Polymeric Amorphous Solid Dispersions: A Review of Amorphization, Crystallization, Stabilization, Solid-State Characterization, and Aqueous Solubilization of Biopharmaceutical Classification System Class II Drugs. J. Pharm. Sci. https://doi.org/10.1016/j.xphs.2015.10.008 Benet, L.Z., 2013. The role of BCS (biopharmaceutics classification system) and BDDCS (biopharmaceutics drug disposition classification system) in drug development. J. Pharm. Sci. 102, 34–42. https://doi.org/10.1002/jps.23359 Best, B.M., Capparelli, E. V, Diep, H., Rossi, S.S., Farrell, M.J., Williams, E., Lee, G., van den Anker, J.N., Rakhmanina, N., 2011. Pharmacokinetics of lopinavir/ritonavir crushed versus whole tablets in children.

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European Directorate for the Quality of Medicines and Healthcare. Gill, D., Spain, M., Edlund, B.J., 2011. Crushing or Splitting Medications: Unrecognized Hazards. J. Gerontol. Nurs. 38, 8–11. https://doi.org/10.3928/00989134-20111213-01 Han Sol, L., Jeong Jun, L., Myeong Gye, K., Ki Taek, K., CHeong Weon, C., Dae Duk, K., Jae Young, L., 2019. sprinkle formulations - a review of commercially available products. Asian J. Pharm. Sci. Janssens, S., Van den Mooter, G., 2009. Review: physical chemistry of solid dispersions. J. Pharm. Pharmacol. 61, 1571–1586. https://doi.org/10.1211/jpp.61.12.0001 Leuner, C., Dressman, J., 2000. Improving drug solubility for oral delivery using solid dispersions. Eur. J. Pharm. Biopharm. 50, 47–60. https://doi.org/10.1016/S0939-6411(00)00076-X Lipinski, C., 2002. Poor aqueous solubility - An industry wide problem in drug discovery. Am. Pharm. Rev. 5, 82–85. Mercovich, N., Kyle, G.J., Naunton, M., 2014. Safe to crush? A pilot study into solid dosage form modification in aged care. Australas. J. Ageing 33, 180–184. https://doi.org/10.1111/ajag.12037 Newman, A., 2015. Pharmaceutical Amorphous Solid Dispersions, Pharmaceutical Amorphous Solid Dispersions. Paparella, S., MSN, R., PA, H., 2010. Identified Safety Risks With Splitting and Crushing Oral Medications. J. Emerg. Nurs. 36, 156–158. https://doi.org/10.1016/j.jen.2009.11.019 Salmon, D., Pont, E., Chevallard, H., Diouf, E., Tall, M.L., Pivot, C., Pirot, F., 2013. Pharmaceutical and safety considerations of tablet crushing in patients undergoing enteral intubation. Int. J. Pharm. 443, 146–153. https://doi.org/10.1016/j.ijpharm.2012.12.038 Sastry, S.V., Nyshadham, J.R., Fix, J.A., 2000. Recent technological advances in oral drug delivery - A review. Pharm. Sci. Technol. Today 3, 138–148. https://doi.org/10.1016/S1461-5347(00)002479 Shah, V.P., Lesko, L.J., Fan, J., Fleischer, N., Handerson, J., Malinowski, H., Makary, M., Ouderkirk, L., Bay, S., Sathe, P., Singh, G.J.P., Iillman, L., Tsong, Y., Williams, R.I., 1997. FDA guidance for industry 1 dissolution testing of immediate release solid oral dosage forms. Dissolution Technol. https://doi.org/10.14227/DT040497P15 Thayer, A.M., Houston, C., 2010. Finding solutions. Custom manufacturers take on drug solubility issues to help pharmaceutical firms move products through development. Chem. Eng. News 88, 13–18. Van den Mooter, G., 2012. The use of amorphous solid dispersions: A formulation strategy to overcome poor

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Discov.

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Technol.

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https://doi.org/10.1016/j.ddtec.2011.10.002 Zhang, J., Han, R., Chen, W., Zhang, W., Li, Y., Ji, Y., Chen, L., Pan, H., Yang, X., Pan, W., Ouyang, D., 2018. Analysis of the Literature and Patents on Solid Dispersions from 1980 to 2015. Molecules 23, 1–19. https://doi.org/10.3390/molecules23071697 Page 20 of 29

Credit author statement: Timothy Pas: data collection, data processing, writing, editing Selam Verbert: data collection, writing, reviewing Bernard Appeltans: reviewing Guy Van den Mooter: conceptualization, writing, reviewing, editing

Page 22 of 29

Nurse

- Amorphous solid dispersions

Enabling Formulations Powder

Suspended in Liquid

or

Hospital Pharmacist

= Mechanical manipulation

Crushing

Swallowing difficulties

Hospitalized Patient

Question(s) Time Uncrushed Formulation Crushed Formulation → Resulting in faster dissolution, higher Cma x and hence early and rapid nucleation and precipitation of the poorly soluble drug? Crushed Formulation → Crushing resulting in crystallization and hence nucleation and precipitation of the poorly soluble drug from the start of dissolution?

Same Performance of enabling formulations before and after crushing? Intraluminal Concentration

In-vitro drug-release testing in nonbiorelevant and biorelevant media showed: → Significant differences in dissolution He Hence, → Crushing enabling formulations might lead to non-equivalence! → Further investigation in humans is needed!

Observations

* Icons from flaticon.com

Figure 1:

Amorphous Solid Dispersions Stability v.s. Solubility Plot – Crystalline and Amorphous Drugs

Generalized Mechanism of Drug Supersaturation Generation and Maintenance During the Dissolution of Amorpous Drugs and ASDs

Solubility

Drug Concentration

Supersaturated (ASD) drug during dissolution

Amorphous drug maximum solubility in presence of polymer

Supersaturated (amorphous) drug during dissolution

Nucleation/Crystal growth

Amorphous drug maximum solubility

Polymer induced supersaturation (metastable)

Crystalline drug equilibrium solubility in presence of polymer

Polymer induced solubilizing effect (stable)

nucleation

Crystal growth

Crystalline drug equilibrium solubility

Stability Time

Same Performance of enabling formulations before and after crushing? Intraluminal Concentration

Legend: Uncrushed Formulation Crushed Formulation → Resulting in faster dissolution, higher Cma x and hence early and rapid nucleation and precipitation of the poorly soluble drug? Crushed Formulation → Crushing resulting in crystallization and hence nucleation and precipitation of the poorly soluble drug from the start of dissolution? Time

Figure 2:

Etravirine Posaconazole

Itraconazole

Ritonavir

Page 23 of 29

Figure 3:

Solubility Screening - Influence of Excipients Solubility Etravirine

0,010 .

. 0,008 0,006 .

. 0,002

Pure medium +1.5% HPMC +3% HPMC +6% HPMC +5% Sucrose +5% PEG 20000 +Mixture

. 0,006 . 0,005 . 0,004

Concentration (mg/mL)

Concentration (mg/ml)

Solubility Itraconazole Pure medium +0.1% HPMC +1% HPMC +2.5% HPMC +5% HPMC

. 0,012

. 0,003 . 0,002 0,001 .

. 0,000

0,000 . SGF pH 1.2

pH 6.8

FaSSGF

SGF pH 1.2

FaSSGF + FaSSIF

pH 6.8

Solubility Posaconazole

FaSSGF + FaSSIF

Solubility Ritonavir

0,1 .

0,007 . 0,006 . 0,005 . 0,004 . 0,003 .

Pure medium +5% PEG 4000 +5% PVP-VA +1% PVP-VA +2.5% HPMC +1% HPC +0.1% PS 80 +Mixture

0,2 .

Concentration (mg/mL)

Pure medium +5% PVA +5% PEG 4000 +1% HPC +1% HPMC-AS +Mixture

0,2 .

Concentration (mg/ml)

FaSSGF

Itraconazole

Etravirine

0,06 . 0,05 . 0,04 . 0,03 . 0,02 .

0,002 .

0,01 .

0,001 . 0,000 .

0,00 . SGF pH 1,2 .

pH 6.8

FaSSGF

FaSSGF + FaSSIF

SGF pH 1.2

pH 6.8

Posaconazole

FaSSGF

FaSSGF + FaSSIF

Ritonavir

Figure 4: 2-stage QC set-up

2-stage biorelevant set-up

Intelence®

Intelence®

FaSSGF . 0,03

0,0003 .

SGF

Intact Ground Limit of quantification

pH 6.8

0,0002 .

. 0,0001

Concentration (mg/mL)

Concentration (mg/mL)

0,0004 .

FaSSGF + FaSSIF Intact Ground Solubility FaSSGF + FaSSIF Limit of quantification

. 0,02

0,01 .

* . 0,00

. 0,0000

0

100

200

Time (min)

300

0

100

200

300

Time (min) n=5 at t= 5 min for grinded Intelence® tablet & at t=300 min for intact Intelence® tablet

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Figure 5: 2-stage QC set-up

2-stage biorelevantset-up

Norvir®

Intact Ground Solubility SGF pH 1.2 Solubility pH 6.8 SGF

pH 6.8

. 0,1

Concentration (mg/mL)

Concentration (mg/mL)

. 0,2

Norvir®

. 0,3

. 0,0

Intact Ground Solubility FaSSGF Solubility FaSSGF + FaSSIF

0,2 .

. 0,1

FaSSGF

FaSSGF + FaSSIF

. 0,0 0

100

200

300

0

100

200

300

Time (min)

Time (min)

n=5 at t= 300 min for Intact Norvir® tablet

n= 5 at t=120 min for grinded Norvir® tablet & at t=180 min for intact Norvir® tablet

Figure 6: 2-stage QC set-up

2-stage biorelevant set-up

Noxafil®

Noxafil®

. 0,08

Intact Ground Solubility SGF pH 1.2 Solubility pH 6.8

. 0,22

SGF

pH 6.8

. 0,20

. 0,010

Intact Ground Solubility FaSSGF Solubility FaSSGF + FaSSIF

. 0,06

Concentration (mg/mL)

Concentration (mg/mL)

. 0,24

. 0,04

. 0,02

. 0,005

FaSSGF + FaSSIF FaSSGF

. 0,000

. 0,00 0

100

200

Time (min) n=1 at t= 300 min for intact Noxafil® tablet

300

0

100

200

Time (min) n=5 at t=140 min for intact Noxafil® tablet

Page 25 of 29

300

Figure 7: 2-stage QC set-up

2-stage biorelevant set-up

Sporanox®

Sporanox®

Intact Ground Solubility SGF pH 1.2 Solubility pH 6.8

. 0,05

0,00015

Intact Ground Solubility FaSSGF Solubility FaSSGF + FaSSIF

. 0,04

Concentration (mg/mL)

0,00030

Concentration (mg/mL)

Concentration (mg/mL)

. 0,10

. 0,02

FaSSGF

FaSSGF + FaSSIF

0,00000 100

200

300

Time (min)

SGF

pH 6.8 . 0,00

. 0,00 0

100

200

Time (min)

300

0

100

200

300

Time (min)

n = 5 at t= 50 min for intact Sporanox® pellets

Page 26 of 29

Table 1: Sporanox® Itraconazole

Intelence® Etravirine

Noxafil® Posaconazole

Norvir® Ritonavir

Physicochemical properties of the drug

BCS class II drug MW (g/mol): 705.6 IB: weakly basic Log P: 6.2 Log D: 7.3 pKa: 2 and 3.7

BCS class IV drug MW (g/mol): 435.2 IB: weakly basic Log P: 5.5 Log D: pKa: 4.1 and 12.5

BCS class II drug MW (g/mol): 700.8 IB: weakly basic Log P: 4.7 Log D: 5.4 pKa: 3.6 and 4.6

BCS class II drug MW (g/mol): 720.9 IB: weakly basic Log P: 3.9 Log D: 4.3 pKa: 2.8 and 13.7

Excipients

100 mg tablets: - HPMC 2910 5mPa.s - PEG 20000 - Sucrose beads - Titanium dioxide - Indigo carmine - Gelatin - Erythrosine

200 mg tablets: - HPMC - (Silicified) MCC - Colloidal anhydrous silica - Croscarmellose sodium - Magnesium stearate

100 mg tablets: - HPMC - AS - MCC - Hydroxypropyl cellulose - Silica dental type - Croscarmellose sodium - PVA - Magnesium stearate - PEG 3350 - Titanium dioxide - Talc - Iron oxide yellow

100 mg tablets: - PVP - VA - Sorbitan laurate - Calcium hydrogen phosphate, anhydrous - Sodium stearyl fumarate - HPMC - Titanium dioxide - Macrogols - HPC - Talc - Colloidal anhydrous silica - PS 80

Dose strength Dosage form Crushing

100 mg Capsules No

25, 100 or *200 mg Dispersible tablets Yes

100 mg Gastro-resistant tablets No

100 mg Film-coated tablets No

Drug

*Dose strength used MW: Molecular weight IB: Ionization behavior Log D: Logarithmic distribution coefficient Log P: Logarithmic partition coefficient MCC: Microcrystalline cellulose HPC: Hydroxypropyl cellulose PVA: Polyvinyl alcohol PS 80: polysorbate 80

Page 27 of 29

Table 2:

Type vessels

QC dissolution method 900 mL SGF (pH 1.2) followed by addition of an appropriate amount of 0.5 M Na3PO4 solution Conventional large vessels

Biorelevant dissolution method 50 mL FaSSGF followed by addition of 150 mL FaSSIF Customized small vessels

Stirring rate (rpm)

75

50

Temperature (°C)

37

37

Sample volume (mL) Time points (min)

1.5

1.5

Dissolution media

5, 15, 30, 50, 60, 70, 90, 120, 140, 180, 240 and 5, 15, 30, 50, 60, 70, 90, 120, 140, 180, 240 and 300 300

Table 3: f2-factors of final conditions 50 RPM (S) vs. 50 RPM (C)

50 RPM (S) vs. 75 RPM (C)

50 mL vs. 900 mL

73.10

79.91

200 mL vs. 955 mL

81.44

92.78

Page 28 of 29

Declaration of interests

‫ ܈‬The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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