Ability of gelatin and BSA to stabilize the supersaturated state of poorly soluble drugs

Accepted Manuscript ABILITY OF GELATIN AND BSA TO STABILIZE THE SUPERSATURATED STATE OF POORLY SOLUBLE DRUGS Timothy Pas, Alina Struyf, Bjorn Vergauwen, Guy Van den Mooter PII: DOI: Reference:

S0939-6411(18)30575-7 https://doi.org/10.1016/j.ejpb.2018.08.003 EJPB 12852

To appear in:

European Journal of Pharmaceutics and Biopharmaceutics

Received Date: Revised Date: Accepted Date:

30 April 2018 23 July 2018 8 August 2018

Please cite this article as: T. Pas, A. Struyf, B. Vergauwen, G. Van den Mooter, ABILITY OF GELATIN AND BSA TO STABILIZE THE SUPERSATURATED STATE OF POORLY SOLUBLE DRUGS, European Journal of Pharmaceutics and Biopharmaceutics (2018), doi: https://doi.org/10.1016/j.ejpb.2018.08.003

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ABILITY OF GELATIN AND BSA TO STABILIZE THE SUPERSATURATED STATE OF POORLY SOLUBLE DRUGS Timothy Pasa, Alina Struyfa, Bjorn Vergauwenb, and Guy Van den Mootera* a

Drug Delivery and Disposition, KU Leuven, Department of Pharmaceutical and Pharmacological Sciences, Campus Gasthuisberg ON2, Herestraat 49 b921, 3000 Leuven, Belgium b Rousselot bvba, Expertise center, Meulestedekaai 81, 9000 Gent, 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

Page 1 of 49

Abstract Gelatin and bovine serum albumin (BSA), two readily available biopolymers, were examined for their effect on solubility and supersaturation of drugs because of their capacity to interact with drugs (e.g. via hydrogen bonding, van der Waals or electrostatic interactions, etc.). Carbamazepine, cinnarizine, diazepam, itraconazole, nifedipine, indomethacin, darunavir (ethanolate), ritonavir, fenofibrate, griseofulvin, ketoconazole and naproxen were selected accordingly as twelve structurally different model BCS Class II drugs. All selected drugs were evaluated for solubility and supersaturation in presence and absence of these two biopolymers in four media (purified water, FaSSIF, FaSSGF and FeSSIF) by means of the shake flask method for 48h and solvent shift induced supersaturation, respectively. In ca. 75% of the supersaturation experiments with these two biopolymers, drug concentrations significantly different (p > 0.05) from solubility were observed with supersaturation factors (SF) varying between 1.28 and 7.89 (p ≤ 0.05) and between 1.16 and 20.51 (p ≤ 0.01). In order to make an estimation on the relevance of these results, a comparison with three commonly used (semi-) synthetic polymers (HPMC, PVP and PVPVA) was included in purified water. This showed that both biopolymers were at least as efficient as the (semi-) synthetic polymers in sustaining induced supersaturation as in ten out of twelve API comparable results were obtained. Keywords: solubility, supersaturation, protein, gelatin, bovine serum albumin (BSA), biopolymers

Page 2 of 49

Chemical compounds with PubChem CID reference: Chemical compound Carbamazepine Cinnarizine Darunavir Diazepam Fenofibrate Griseofulvin

PubChem CID 2554 1547484 Ethanolate: 23725083 3016 3339 441140

Chemical compound Indomethacin Itraconazole ketoconazole naproxen nifedipine ritonavir

PubChem CID 3715 55283 456201 156391 4485 392622

Non-common abbreviations: … Abbreviations common in the field: - API – Active Pharmaceutical Ingredient - ASD – amorphous Solid Dispersion(s) - AUC – Area Under the Curve - BCS – Biopharmaceutics Classification System - BSA – Bovine Serum Albumin - CI95% – 95%-confidence interval - df – degrees of freedom - DMF – Dimethylformamide - DMSO – dimethylsulfoxide - DS (DSs, DS’s) – Degree of Supersaturation(s) - DSC – Differential Scanning Calorimetry - FaSSGF – Faster State Simulated Gastric Fluid - FaSSIF – Faster State Simulated Intestinal Fluid - FeSSIF – Fed State Simulated Intestinal Fluid - GI-tract – Gastro Intestinal tract - HPMC – Hydroxypropyl methyl cellulose - LOQ – Limit of Quantification - LOD – Limit of Detection - NMP – N-methyl pyrolidone - p – p-value (used in statistical hypothesis testing) - PP – PolyPropylene - PVP – Polyvinylpyrrolydon = Kollidon 30 - PVPVA – Polyvinylpyrrolydon vinylacetate = Copovidone - R – Resistance - SF (SFs) – Supersaturation Factor(s) - SS – SuperSaturation - XR(P)D – X-Ray (Powder) Diffraction - α – level of significance – Mean

Abbreviation uncommon in the field: - /

Page 3 of 49

1. Introduction The search for innovative technologies to tackle the contemporary problem of poor aqueous drug solubility and dissolution rate is of great interest to today’s pharmaceutical industry [1] due to emerging BCS Class II (and IV) drug candidates [2,3,4,5]. Amongst the diversity of available formulation strategies, amorphous solid dispersions (ASD) are gaining momentum and are considered to be very promising [6,7,8]. Amorphous solid dispersions (ASD) are interesting because they are composed of an inert carrier (until now mostly (semi-) synthetic polymers) in which poorly soluble drugs are dispersed at molecular level. Crystallization of the dispersed drugs can be retarded and even avoided due to formation of inter-molecular interactions between the drug and the carrier, exhibition of anti-plasticizing effect(s) by the carrier, creation of a physical barrier to the nucleation/crystallization process of the drug by the carrier due to increased viscosity (decreased mobility) and/or reduction of Gibbs energy [7,9,10]. Hence, the presence of the carrier results in reduction of structural relaxation, nucleation and crystal growth [7,11,12]. Carrier selection thus plays a critical role in the field of solid dispersions as it largely determines their overall performance. Ideally, a carrier must be able to stabilize the molecularly dispersed state of the API (often above the thermodynamic solubility) whilst simultaneously enhancing the dissolution rate and maintaining the supersaturated state in the GI-tract to have adequate

driving

force

for

absorption

[9,10,13,14,15].

Today,

Accolate®,

Accupril®,

Advagraf®/Astagraf XL®, Afeditab® CR, Ceftin®, Certican®/Zortress®, Cesamet®, Crestor®, Fenoglide®, Gris-PEG®, Incivek®/Incivo®, Intelence®, Isoptin® SR-E 240, Kaletra®, Kalydeco®, Lozanoc®, Mesulid® fast, Nimotop®, Nivadil®, Norvir®, Noxafil®, Prograf®/LCP-Tacro®, Rezulin®, Sporanox®/Onmel®, Thomaflex Meltrex®, Viracept®, and Zelboraf® are examples of solid dispersion formulations that already received market authorization [5, 7,16,17,18,19]. Currently, both in marketed and investigational ASD formulations, diverse (semi-) synthetic polymers like HPMC, PVP and PVPVA have predominantly been explored. However, we recently

Page 4 of 49

demonstrated the potential of gelatin 50PS as carrier in the formulation of amorphous solid dispersions [20], likely to be attributed to the biopolymers’ promising capacity for reversible drug binding which is already widely described in literature for plasma proteins [21,22,23] and which can also be derived from their structures itself (e.g. rich of hydrogen bond forming sites). Biopolymers are also in most cases “greener” alternatives to the currently most used (semi-) synthetic ones [24]. Other biopolymers so far reported in literature for formulation of solid dispersions, are egg albumin [25], casein [26], whey proteins [27] and bovine serum albumin (BSA) [28,29]. Also noteworthy are some papers reporting on co-amorphous formulations of drug(s) with amino acids [30,31,32,33]. ASD formulations of the above quoted biopolymers were obtained by means of technologies like e.g. the kneading method [25,26], coprecipitation [26], freeze-drying [20,28,29], spray drying [26,28,29] and vibrational ball milling or cryomilling [30,31,32,33]. Depending on the preparation method, certain pitfalls might be present for biopolymers as discussed in our previous work on gelatin [20]. Because the introduction of biopolymers in the field of ASDs happened relatively recently, information on their potential to influence solubility and supersaturation effects of poorly soluble drugs is still scarce in comparison to the earlier introduced (semi-) synthetic polymers. Therefore, the aim of the present study is to further explore the capability of gelatin and BSA to influence and maintain supersaturation of the same twelve BCS Class II model drugs (carbamazepine, cinnarizine, diazepam, itraconazole, nifedipine, indomethacin, darunavir, ritonavir, fenofibrate, griseofulvin, ketoconazole and naproxen (see Figure 1)) in four different media (purified water, FaSSIF, FeSSIF and FaSSGF). In addition, we will compare the solubilizing and supersaturating potential of gelatin and BSA with commonly used polymers like PVP, HPMC and PVPVA.

Page 5 of 49

2. Materials and Methods 2.1. Materials Carbamazepine (PubChem CID: 2554) and diazepam (PubChem CID: 3016) were obtained from SA Fagron NV (Waregem, Belgium), indomethacin (PubChem CID: 3715) and ketoconazole (PubChem CID: 456201) from ABC Chemicals (Wauthier-Braine, Belgium), griseofulvin (PubChem CID: 441140) and naproxen (PubChem CID: 156391) from Certa (Braine-l’Alleud, Belgium), cinnarizine (PubChem CID: 1547484) from Sigma-Aldrich (Rajasthan, India), darunavir (ethanolate, PubChem CID: 23725083) from Cilag AG (Schaffhausen, Switserland), fenofibrate (PubChem CID: 3339) from Hangzhou Dayangchem Co. (Hangzhou City, China), nifedipine (PubChem CID: 4485) from Indis (Aartselaar, Belgium), ritonavir (PubChem CID: 392622) from Pharmidex (London, UK) and itraconazole (PubChem CID: 55283) from Janssen Pharmaceutica NV (Beerse, Belgium). Tetrabutylhydrogensulfate, methanol, dimethyl formamide (DMF) 99.5% and dimethyl sulfoxide (DMSO) 99.9% were obtained from ACROS Organics (Geel, Belgium), acetonitrile from Fisher Scientific (Leicestershire, U.K.), citric acid monohydrate, phosphoric acid 85% and anhydrous sodium acetate from Chemlab NV (Zedelgem, Belgium). Sigma-Aldrich supplied N-methyl-2-pyrrolidone (NMP) (Gillingham, UK) and mono- (monohydrate) (Steinheim, Germany) and dibasic sodium phosphate (Gillingham, UK). NaOH pellets were produced by BDH Laboratory Supplies (Poole,U.K.). Hydrochloric acid and (glacial) acetic acid were obtained from VWR (Fontenay-sous-Bois, France). Biorelevant (London, U.K.) supplied FaSSIF/FeSSIF/FaSSGF instant powder. Water was purified with a Maxima system (Elga Ltd., High Wycombe Bucks, U.K.) resulting in purified water (pH = 6.05 ± 0.04; R > 18 Ohm). Bovine serum albumin (BSA), heat shock fraction, pH 7, ≥ 98%, was obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).

Page 6 of 49

Gelatine 50PS, supplied by Rousselot bvba (Gent, Belgium), was manufactured using an acidpretreatment (Type A process) and is characterized by having a Bloom value of 56 grams, a viscosity at 6.67% (w:w) of 1.75 mPa.s, and an isoelectric point of 8.5. Hydroxypropyl methyl cellulose (HPMC 5 mPas) was obtained from Colorcon (Idstein, Germany), whilst Kollidon 30 (= Polyvinylpyrrolydon = PVP) and Kollidon VA 64 (= Polyvinylpyrrolydon vinylacetate = PVPVA) were received from BASF (Ludwigshafen, Germany). 2.2. Preparation of media for solubility and supersaturation studies 2.2.1. Non-biorelevant media Purified water as such (pH = 6.05 ± 0.04; R > 18 Ohm), purified water containing 1 % (w/V) of gelatin 50PS (pH = 4.98 ± 0.01) and purified water containing 1% (w/V) of BSA (pH = 7.18 ± 0.01) were selected as non-biorelevant media for solubility and supersaturation testing (see 2.3 and 2.4). The 1% (w/V) gelatin 50PS solution was prepared by dissolving gelatin 50PS by stirring and heating (≤ 120°C) until a clear solution was obtained. This solution was cooled to ambient temperature before use. For dissolving BSA, only stirring was used. In addition, aqueous solutions containing 1 % (w/V) HPMC (pH = 4.74 ± 0.01), 1 % (w/V) PVP (pH = 4.00 ± 0.01) and 1 % (w/V) PVPVA (pH = 4.30 ± 0.01) were prepared with the same purified water as mentioned above and in the same way as described for BSA-containing media. 2.2.2. Biorelevant media Solubility and supersaturation tests were also performed in purified water, fasted state simulated intestinal fluid (FaSSIF), fed state simulated intestinal fluid (FeSSIF) and fasted state simulated gastric fluid (FaSSGF) as well as in these biorelevant media supplemented with 1% (w/V) of gelatin 50PS or BSA (see 2.3 and 2.4). The composition and characteristics of the biorelevant media are provided in Appendix A (Table A.1).

Page 7 of 49

These media were prepared by primarily weighing the required quantities of FaSSIF/FeSSIF/FaSSGF (biorelevant) powder, NaCl, monobasic sodium phosphate monohydrate, NaOH and (glacial) acetic acid. Secondly, purified water (1/3th of the final volume) was used to dissolve these substances except for the FaSSIF/FeSSIF/FaSSGF (biorelevant) powder. Subsequently, only for the 1% (w/V) gelatin 50PS and 1% (w/V) BSA containing media, 3% (w/V) stock solutions of these two biopolymers in purified water were prepared. From these stock solutions, 1/3th of the final required volume of the medium was taken and added to the dissolved substances of the previous step in case a 1% (w/V) biopolymer containing medium was prepared. In a next step, both for biopolymer- as nonbiopolymer-containing media, purified water was used to reach 90-95% of the final volume of the medium. Thereafter, media were pH-adjusted with 1M NaOH and/or 1M HCl (pH 6.5 for FaSSIF, pH 5.0 for FeSSIF and pH 1.6 for FaSSGF). After pH-adjustment, FaSSIF/FeSSIF/FaSSGF (biorelevant) powder was added and purified water was added until the final volume was reached. Finally, all media were left untouched for at least 2 hours before use (only necessary for FaSSIF). Media were than kept at ambient temperature and used within 48 hours following their preparation. (Scheme for preparation can be found in Appendix B) 2.3. Solubility studies Thermodynamic solubility of all drugs, in different media and in absence or presence of gelatin 50PS or BSA (See Appendix A, Table A.2), was determined in triplicate in 10 mL glass test tubes by using the shake-flask method for 48 hours. After 48 hours and 15 minutes of centrifugation at 20238 g, the supernatant was used as such or diluted with DMSO, DMSO-H2O or NMP-H2O mixtures for HPLC-analysis (see 2.5). In addition, the same solubility experiment was repeated for the non-biorelevant aqueous media containing 1% (w/V) HPMC, 1% (w/V) PVP and 1% (w/V) PVPVA (See Appendix A, Table A.3) with the only difference that in this case samples were diluted with mobile phase instead of DMSO, DMSOH2O or NMP-H2O mixtures before HPLC-analysis.

Page 8 of 49

2.4. Supersaturation studies Supersaturation of all twelve selected compounds was investigated, in presence of 1% (w/V) gelatin 50PS or 1% (w/V) BSA, using the solvent-shift method and additionally the temperature-shift method for cinnarizine. The temperature-shift method was applied as follows: residual undissolved cinnarizine in the organic solvent was dissolved by slightly heating the solution to enhance and increase solubility for a short period after which the solvent-shift method could be carried out with the heated organic solvent (see next paragraphs). Supersaturation experiments were performed, in triplicate, in each of the 8 media containing 1% (w/V) gelatin 50PS or 1% (w/V) BSA. The experimental set up was as follows: Experiments were carried out in 50 mL glass beakers, containing a magnetic stirrer. The magnetic stirrer device was operated at ca. 370 rpm. Before starting the actual experiment, beakers were prefilled with the desired medium, which corresponded to a volume of 20 mL minus the volume of stock solution to be added (see further). Based on the preliminary acquired equilibrium solubilities determined in “2.3. Solublity studies”, stock solutions in DMSO, NMP or DMF of the twelve various drugs were prepared to induce an initial degree of supersaturation (DS) equivalent to 5, 10 or 20 depending on the compound. From these stock solutions a maximum of 400 µL was added to the prefilled beakers with medium, corresponding to a final organic solvent content ≤ 2%. After adding these stock solutions, 1 mL samples were taken after 15, 30, 45, 60, 90 and 120 minutes and subsequently centrifuged for 15 minutes at 20238 g in an Eppendorf AG centrifuge 5424 (Hamburg, Germany). After centrifugation, supernatants of the samples were properly diluted with DMSO, DMSO-H2O or NMP-H2O mixtures for HPLC-analysis. The extent of dilution and selection of dilution mixture was API dependent. Samples for HPLC-analysis were vortexed with a VortexGenie 2 from Scientific Industries (Bohemia, USA) before analysis. In addition, the same supersaturation experiment was repeated for the non-biorelevant aqueous media containing either no polymer, 1% (w/V) HPMC, 1% (w/V) PVP or 1% (w/V) PVPVA with the only

Page 9 of 49

difference that in this case samples were diluted with mobile phase instead of DMSO, DMSO-H2O or NMP-H2O mixtures before HPLC-analysis. At the end of each supersaturation experiment, the precipitate was collected by centrifugation (30 min at 2880 g in polypropylene (PP) falcon tubes in an Eppendorf centrifuge 5804 R from Eppendorf AG (Hamburg, Germany)) and analyzed with XR(P)D. After acquisition, supersaturation data was processed together with the solubility data from 2.3. as concentration-time profiles. To estimate of the overall supersaturation effect of these drugs for the different media in function of time, supersaturation factors (SFs) for the time range 15 min – 120 min were calculated based on mean AUC values (Eq. 1): SF =

(Eq. 1)

In addition, it was statistically evaluated for the 15 min – 120 min time region, by calculating 95% confidence intervals (CI95%), if the mean AUC’s of the supersaturation tests differed significantly from those of solubility testing or not. This evaluation contributed to the significance of the calculated SF. Because triplicate measurements were used, n < 30, the deviation from the normal distribution is more leptokurtic than for n ≥ 30. Therefore, the following equation (Eq. 2) was used to determine CI95% [34,35,36].

(Eq. 2)

Where

corresponds to the mean AUC of a triplicate measurement,

to the t-

value obtained from a unilateral t-table at the α/2 = 0.025 level with n-1 degrees of freedom (df), to the standard deviation and n to the number of experiments to determine the mean AUC. As measurements were only performed in triplicate, n corresponded to 3 and

= 4.303

was used for calculations which is larger than when n is sufficiently large (n ≥ 30 → t close to 1.960). If in the end CI95% of the mean AUC’s of the supersaturation tests and those of solubility testing did Page 10 of 49

not show overlap, their difference was considered statistically significant [34,35,36]. This is equivalent to performing an unpaired t-test at the 5%-level, where p-values < 0.05 are considered statistically significant. 2.5. HPLC analysis All drug concentrations (solubility and drug release studies) were determined by HPLC-analysis using either a VWR HITACHI Chromaster system (consisting of a 5410 UV detector, a 5310 column oven, a 5260 auto sampler and a 5160 pump) or a Merk-Hitachi LaChrom system (consisting of a D7000 interface, a L-7420 UV-VIS detector, a L-7200 auto sampler and a L-7100 pump). Data acquisition was performed using Chromaster System Manager Software (version 1.1) for the VWR HITACHI Chromaster system and Merck LaChrom D-7000 system Manager Software (version 4.1) for the Merk-Hitachi LaCrom system. For each of the twelve compounds a validated isocratic HPLCmethod was constructed and further used. All chromatographic parameters are listed in Appendix C. 2.6. Solid state analysis of precipitates X-ray (powder) diffractometry (XR(P)D) analysis of collected precipitates was conducted within 24 hours following collection using an automated X’pert PRO diffractometer (PANalytical, Almelo, The Netherlands) with a Cu tube (Kα1 λ = 1.5418 Å), and a generator set at 45 kV and 40 mA. The measurements were performed at room temperature, in transmission mode using Kapton® Polyimide Thin-films (PANalytical, USA). The selected experimental settings included a continuous scan mode from 4° to 40° 2θ with 0.0167° step size and 400 s counting time. 2.7. Dialysis of gelatin 50PS Dialysis was performed as follows: 70 mL of a 10 % (w/V) gelatin 50PS solution in purified water was transferred to a ThermoScientific slide-A-lyzer® 3.5K dialysis cassettes G2 (3500 MWCO) obtained from Thermo Scientific (Rockford, USA) for dialysis. Actual dialysis was performed by subsequently hydrating the membrane of the dialysis cassette, filling it with 10 % (w/V) gelatin 50PS

Page 11 of 49

solution and dialyzing it by immersing it in 5L purified water which was stirred. The purified water was replaced twice a day for seven subsequent days. During the last four days, heating until 70°C was also applied to even improve dialysis. In the end, the dialyzed gelatin 50PS solution was collected and lyophilized until dryness.

Page 12 of 49

3. Results and Discussion 3.1. Solubility 3.1.1. Solubility in pure media Knowledge about the twelve drugs’ equilibrium solubility in the different media, with and without the biopolymers, is essential in order to determinine the “true” effect of supersaturation induced by both BSA and gelatin 50PS. All equilibrium solubility data are summarized in Figure 2. These data illustrate the wide diversity in physicochemical characteristics between the different drugs as different observations were made depending on the API or chemical class of APIs. In addition, in order to fully understand following data, pH-dependent charge predictions for all twelve APIs over the full pH-range (1-14) are given in Figure 3 (predicted by using MarvinSketch Software). Indomethacin and naproxen are weak acids whilst cinnarizine, diazepam, itraconazole and ketoconazole are weak bases. Carbamazepine, fenofibrate, griseofulvin and nifedipine are neutral compounds. Although, darunavir and ritonavir are both amphoteric compounds, their behavior can also be described in the same way as for the weak bases as their acidic functions are not deprotonated below pH 11 – 12 which is the case for the different considered media. This classification is summarized in Table 1. Weak acids, such as naproxen and indomethacin, tended to be worst soluble in acidic environment (FaSSGF, see Figure 2, g and j) which can be attributed to their carboxylic acid function (see Figure 1) with pKa ≈ 4.2 and pKa ≈ 3.8, respectively. At pH = 1.60 (gastric environment, FaSSGF) these carboxylic functions will be mostly protonated (see Figure 3, g and j), resulting in the worst solubility of all tested media. Hence, solubility increases for these weak acids when going from gastric medium (FaSSGF, pH = 1.60) towards the more neutral fasted intestinal medium (FaSSIF, pH = 6.50) (see Figure 2, g and j). However, environmental pH is not the only factor influencing solubility for these compounds. FaSSGF, FaSSIF and FeSSIF media also include bile salts and lecithin (see Appendix A, Table A.1), contributing to the presence of micelles that might also add to the drugs’ solubility. Page 13 of 49

Although intuitively a decreasing solubility for FeSSIF (most bile salts), FaSSIF and FaSSGF (less bile salts) would be expected, this was not observed (see Figure 2, g and j). On the contrary, FeSSIF with five times more bile salts than FaSSIF resulted in lower solubility. Presumably, this is attributed to concomitantly occurring pH- and solubilizing-effects that are evident from the predicted Log P and Log D values of these weak acids (Table 2) and their pH-dependency (Figure 2, g and j). These mutually dependent parameters (Log P, Log D and pH dependency) predict Log D values to diminish when deprotonation is more prone. This complex interplay predicts solubilization of these weak acids to be less susceptible in FeSSIF than in FaSSIF due to less deprotonation. As a result, pH seems to be the main driving force for solubility in case of the investigated weak acids (See Figure 3 and Appendix D, Figure D.1). In contrast to the above discussed weak acids, weak bases like cinnarizine, diazepam, itraconazole and ketoconazole in general demonstrated higher solubility values in FaSSGF (pH 1.6) (see Figure 2, b, d, h, and i). Clearly, this is attributed to the occurring high(er) ionization/protonation of their basic functions in more acidic environments with pKa’s of their basic functions of ≈ 2.9 (nitrogen of diazepam), ≈ 8.8 and ≈ 4 (imidazole and piperazine group of ketoconazole), ≈ 2.7 and ≈ 10.3 (piperazine of cinnarizine) and ≈ 2.2 and ≈ 3.1 (triazole and piperazine function of itraconazole) (also see Figure 3). In essence, weak bases in the selected different (bio-)relevant media tent to be most soluble in acidic environment (FaSSGF) as expected, followed by FeSSIF, FaSSIF and purified water with decreasing extent (see Figure 2, b, d, h, and i). Non-ionizable drugs, such as carbamazepine, fenofibrate, griseofulvin and nifedipine (see Figure 1) are found to be pH-independent (see Figure 3, a, e, f and n). Solubility values of these drugs, as would be expected, seemed to be more pronounced when solubilizing effects with bile salts (FaSSGF < FaSSIF < FeSSIF) were also present (see Figure 2, a, e, f and n).

Page 14 of 49

Although more pronounced in FeSSIF, differences between these four “neutral” compounds were observed (see Figure 2). For carbamazepine and griseofulvin the extent of the solubility in FeSSIF compared to their solubility in the other biorelevent media was less pronounced than those for nifedipine and fenofibrate (see Figure 2, a, e, f and n). These differences between “neutral” compounds are probably attributed to their differences in lipophilicity, which are related to their log P / Log D value(s) (See Appendix D (Figure D.1) and Table 2). Darunavir and ritonavir, both amphoteric drugs, behave as weak bases since their very weak acidic functions are deprotonated at a pH higher (≥ 11-12) than those of these biorelevant media (see Figure 3, c and l). As a result, highest solubilities in FaSSGF (pH = 1.60) were observed because of protonation of the basic functions (see Figure 2, c and l). 3.1.2. Solubility in the presence of gelatin 50PS and BSA It was observed for almost all selected drugs that BSA had a positive influence on their solubility (see Figure 2). Reversible drug binding with BSA and/or changing the water structure (“structure breaking”) effects by presence of dissolved BSA are plausible explanations. Drug binding capability of BSA is already known for a long time and extensively described in literature [21,22,23]. Nevertheless, in some cases (darunavir, ketoconazole, …) the BSA-containing media resulted in solubility values significantly below those of the pure medium (See Figure 3c and 3i). Possibly pH-effects could have been in play here because non-biorelevant media were not pH adjusted in the end at a predefined pH (see Appendix A (Table A.2)). Because a 1% BSA solution has a higher pH (7.18 ± 0.01) than purified water (6.05± 0.035), it can be noticed from Figure 3i that ketoconazole is more protonated in purified water than in a 1% BSA containing solution. From this observation the decreased solubility for ketoconazole is plausible to be due to differences in pH. For darunavir on the other hand, this difference in protonation is less pronounced or even non-existent (See Figure 3c). For gelatin 50PS containing media, in contrast, the influence on solubility was less clear since not always an increase in solubility was observed. Of course, as described for BSA, significant drops in Page 15 of 49

solubility occurring in non-biorelevant media for in this case weak acids (indomethacin and naproxen) could possibly also be attributed to differences in ionization (See Figure 3g and 3j). Nevertheless, for some of the other drugs (carbamazepine, darunavir and ritonavir) solubility values were also lower in presence of gelatin 50PS for which differences in pH of the medium could not explain this observation. One possible explanation for this outcome might be attributed to the presence of impurities in this quality of gelatin because it was not pharmaceutical grade quality but food grade (were more impurities are allowed) instead. To make sure if these impurities could have affected solubility, gelatin 50PS was dialyzed to purify it, subsequently lyophilized to dryness and in the end used for solubility testing of carbamazepine and darunavir as model compounds. From this new solubility experiment with dialyzed and lyophilized gelatin 50PS, it was observed that carbamazepine’s solubility in “purified” gelatin 50PS solution did not significantly differ (p > 0.05) from its solubility in purified water (data not shown). However, for darunavir the solubility values were not significantly different from the first series of solubility tests (data not shown) indicating that the drop in solubility is caused by gelatin and not by its impurities.

3.2. Supersaturation maintenance in the presence of gelatin 50PS and BSA Based on solubility experiments, supersaturation screening was carried out pursuing a Degree of Supersaturation (DS) equal to 20, 10 or 5 in all media. A DS as high as possible, but limited to 20, was aimed for since supersaturation is known to be a driving force for precipitation. In order to enable comparison of supersaturation stabilization of a given drug in different media, equal DS per API was installed. As a result, final DS was limited by the best dissolving medium (except for diazepam for which DS in FaSSGF is lower in comparison to other media) (see Appendix E (Table E.1)). Supersaturation factors (SFs) were calculated by dividing the AUC15min-120min of the collected supersaturation values by the AUC15min-120min obtained from the equilibrium solubility in presence of polymer (see Table 3 for SF and Appendix F for concomitant solubility and SS profiles). Hence, the supersaturation effect additional to the biopolymer’s solubilizing capacity was estimated (see Figure Page 16 of 49

4 for clarification). In 25% of these supersaturation experiments, calculated supersaturation factors (SF) were non-significantly different (> 0.05) from solubility. Supersaturation factors varied between 0.40 and 2.58. In all other cases, significant supersaturation was obtained, with SF varying between 1.28 and 7.89 (p ≤ 0.05) and between 1.16 and 20.51 (p ≤ 0.01). In case of BSA the extent of supersaturation was in general larger than with gelatin 50PS. In addition to Table 3, SFs were also calculated against their solubility in absence of biopolymer (See Table 4). These values reflect the total effect of addition of the polymer, which consists of a solubilizing effect and its real supersaturation effect. In general, increased SFs were obtained in this way for most combinations of drugs and media because the polymer solubilizing effect (when present) is also taken into account. The effect of gelatin and BSA on precipitation inhibition appeared to be compound dependent (see Table 3). For some API gelatin 50PS turned out to have the best stabilizing effect whilst BSA performed best in the other cases. In fact, similar results were obtained with inclusion of either 1% gelatin or 1% BSA. The higher solubility values of the API in BSA solution were not accompanied by lower supersaturation factors as can be seen in Table 3. No particular relationship was found between the biomolecule, the media and the drug. Provided that the magnitude of observed supersaturation is the result of a complex interplay between pH (ionization of the API), interactions (van der Waals, electrostatic and hydrogen bonding), inhibition of nucleation, presence of solubilizing effects (bile salts, …), etc., it is difficult to assign well defined properties that explain differences and make it able to really compare these media (purified water, FaSSIF, FaSSGF and FeSSIF). 3.3. Points of attention during supersaturation testing Applying various degree(s) of supersaturation (DS) seemed to have a profound influence on the subsequently calculated supersaturation factors (SF). As a result, lowering the DS for a certain drug in a certain medium led to an increase in its calculated SF for that same medium. Two examples, Page 17 of 49

diazepam in aqueous media and naproxen in FaSSGF, are given in Table 5 to illustrate this phenomenon. The phenomenon of rising SF with decreasing DS is a result of decreased driving force towards precipitation since a lower amount of drug is added to the same volume of medium. Hence, mentioning the applied DS in supersaturation screening is important to be able to extract conclusions from it. For that reason the highest obtainable DS, respectively 20, 10 or 5, was applied to all media for each separate drug to make comparisons between media for the same API possible. A second point of attention is that in some cases droplet formation was observed after addition of the concentrated API-containing organic solutions to the media for supersaturation testing. This effect seemed to be caused by the apparent high viscosity of the highly drug concentrated organic solution which did not mix easily with the media. When observed, it was noticed that these droplets tended to mix gradually over time resulting in increasing concentration-time profiles instead of decreasing ones as normally expected during supersaturation testing. When present, this phenomenon will of course result in an underestimation of the calculated SF. Since this effect was most profound for indomethacin, when adding the organic solvent to aqueous 1% BSA and FeSSIF 1% BSA media, these two cases were further investigated (See Figure 5 and 6). We observed that the effect was less pronounced if the same amount of indomethacin was added by means of a higher volume of a less concentrated organic solution (See Figure 5). Although, further dilution of the added organic phase to lower drug concentration is not feasible as the added volume of organic phase will increase which will influence solubility. In view of this, aiming at a solvent concentration below 2 V/V% of organic solvent is advised (preferably as low as possible). Secondly, we investigated the effect of ca. 10 seconds of homogenization (See Figure 6). This experiment illustrated that after obtaining a more homogeneous system, the more typical decreasing concentration-time profile is observed. Hence, in some cases a homogenization step may need to be considered. A third point of attention is the difficulty to perform SS experiments due to high solubility. Supersaturation experiments for ketoconazole were carried out starting from a DS 5 by dissolving the

Page 18 of 49

drug in DMSO (See Table 7). Since ketoconazole is a weak base, the thermodynamic solubility values in FeSSIF (pH 5) and FaSSGF (pH 1.6) were extremely high (See Figure 2). Difficulties were encountered when dissolving this high amount of drug in an appropriate solvent to attain a proper degree of supersaturation. Even DS 1.5 was tried, however the same issue occurred. No appropriate organic solvent was found meeting the requirement of high solubilizing capacity. Hence, supersaturation tests of ketoconazole in FeSSIF and FaSSGF could not be performed due to its decreased relevance.

3.4. Investigation of collected precipitates The outcome of the XR(P)D measurements of all collected precipitates during supersaturation are summarized in Table 6 (Corresponding XR(P)D profiles can be found in appendix G). It was observed that carbamazepine always tended to precipitate as another polymorph than the starting material. Precipitates of griseofulvin and nifedipine always crystallized in the same modification as the starting material, whereas the precipitates of darunavir (and itraconazole) were always X-ray amorphous. For all other drugs, both crystalline and X-ray amorphous precipitates were detected. Although of great value, solely XR(P)D-screening might not be sufficient to fully characterize the mode of precipitation as it might fail to detect nanocrystallinity when present in small amounts.

3.5. Comparison of the SS potential of gelatin 50PS and BSA with (semi-) synthetic polymers Because the use of biopolymers is emerging as carrier(s) in solid dispersions, their relevance in this field should be addressed compared to widely applied carriers. Hence, comparison of gelatin 50PS and BSA with three (semi-) synthetic polymers (HPMC, PVP and PVPVA) was performed in nonbiorelevant media (purified water with or without 1% biopolymer). Solubility values, observed during testing, are summarized as histogram plots in Figure 7. These data in general comply with the evaluation shown in part 3.1.1.. In addition, it was recognized that in some cases (evidently clear for cinnarizine, fenofibrate, indomethacin and itraconazole) the solubility Page 19 of 49

in presence of 1% BSA was well superior to those in presence of (semi-) synthetic polymers. Possibly, this can be attributed to the exceptional binding capacity of BSA’s well defined high affinity regions for ligand binding (also known as the Sudlow’s site I and site II which are located in sub-domains IIA and IIIA) for drugs [37]. However, this remains to be further investigated. Significant supersaturation was observed in 62.1% of the tests with SF ranging between 1.36 and 4.08 (p ≤ 0.05) and between 1.21 and 18.35 (p ≤ 0.01) (see Table 7, based on results depicted in Appendix H). Along Table 7, SFs were calculated additionally against their solubility in absence of polymer (See Table 8).

The effect of supersaturation induced by presence of 1% gelatin 50PS and 1% BSA was at least comparable to those of the (semi-) synthetic polymers. This is evidently clear if the number of drugs giving rise to significant supersaturation are considered for these different polymers (see Figure 8). Although nothing about the extent of supersaturation is clear from Figure 8, SF shown in Table 3 and 8 are of the same order for all polymers (in purified water). Only in case of carbamazepine and darunavir the (semi-) synthetic polymers outperformed the biopolymers. As a result, both biopolymers addressed here seem to be excellent competitors when stabilization of the SS is required and hence display attractive characteristics to be used as a carrier in solid dispersions.

Page 20 of 49

4. Conclusion Gelatin 50PS and BSA as biopolymers have shown to be at least comparable to more commonly used (semi-) synthetic polymers like HPMC, PVP and PVPVA in maintaining SS. Hence, biopolymers in general could be valuable alternatives to these (semi-) synthetic polymers if maintenance of SS is concerned. Additionally, the performed solubility experiments show-cased the complex interplay that exists between ionization, micellar solubilization, ionic strenght, etc. which all might exert an effect on solubility depending on the drugs’s characteristics. Finally, it was also observed that some points of attention have to be considered whilst performing SS experiments. First of all, DS-dependency was observed which resulted in increased SS with decreasing DS. For that reason, if possible, DS = 20 was pursued (generally accepted in literature) to not overestimate the effect. Secondly, occurrence of droplets is possible if highly concentrated drug solutions are added during the SS experiment resulting in viscous droplet formation from which drugs are slowly released towards the SS medium resulting in an underestimation of SS. More diluted drug solutions and/or homogenization seemed reduce this problem. Lastly, the relevance of SS experiments in highly drug soluble media has to be addressed. In general, an underestimation is more likely to be observed than an overestimation. As a result, this study in combination with our preceeding work [20] seems to confirm the potential of gelatin 50P and BSA as alternative carriers to formulate amorphous solid dispersions.

Page 21 of 49

Acknowledgements We would like to acknowledge Rousselot N.V. (Meulestedekaai 81, 9000 Gent, Belgium) for their financial support.

Page 22 of 49

Figure captions: Figure 1: Chemical structures of carbamazepine, cinnarizine, diazepam, itraconazole, nifedipine, indomethacin, darunavir, ritonavir, fenofibrate, griseofulvin, ketoconazole and naproxen. Created with MarvinSuite software (ChemAxon). → 2 column figure Figure 2: Solubilities of (a) carbamazepine, (b) cinnarizine, (c) darunavir, (d) diazepam, (e) fenofibrate, (f) griseofulvin, (g) indomethacin, (h) itraconazole, (i) ketoconazole, (j) naproxen, (k) nifedipine and (l) ritonavir in aqueous media, FaSSIF, FeSSIF and FaSSGF, either as such or including 1% gelatin or 1% BSA as biopolymer determined by the shake-flask method for 48h to reach equilibrium. Black bars = solubility in pure medium, Red bars = solubility in medium containing 1% gelatin 50PS and Blue bars = solubility in medium containing 1% BSA. In addition, standard deviation bars are also added. Values indicated on solubility bar-plots are the corresponding solubility values of the corresponding bars. → 2 column figure (both parts together or separate, depending on preference) Figure 3: pH-dependent charge predictions over the full pH-range (1-14) of (a) carbamazepine, (b) cinnarizine, (c) darunavir, (d) diazepam, (e) fenofibrate, (f) griseofulvin, (g) indomethacin, (h) itraconazole, (i) ketoconazole, (j) naproxen, (k) nifedipine and (l) ritonavir (predicted by using MarvinSketch Software). → 2 column figure (both parts together or separate, depending on preference) Figure 4: Illustration of AUC integration to be used for SF calculation. → 2 column figure Figure 5: Effect of less concentrated indomethacin solution added to 1% BSA in purified water solution in case droplet formation was observed. → 1 column figure Figure 6: Effect of homogenization of indomethacin solution added to 1% BSA in FeSSIF solution in case droplet formation was observed. → 1 column figure Figure 7: Solubilities of (a) carbamazepine, (b) cinnarizine, (c) darunavir, (d) diazepam, (e) fenofibrate, (f) griseofulvin, (g) indomethacin, (h) itraconazole, (i) ketoconazole, (j) naproxen, (k) nifedipine and (l) ritonavir in aqueous media, either as such or including 1% HPMC, 1% PVP or 1% Page 23 of 49

PVPVA as polymer determined by the shake-flask method for 48h to reach equilibrium. Black bars = solubility in pure medium, Red bars = solubility in medium containing 1% gelatin HPMC, Blue bars = solubility in medium containing 1% PVP and Purple bars = solubility in medium containing 1% PVPVA. In addition, standard deviation bars are also added. Values indicated on solubility bar-plots are the corresponding solubility values of the corresponding bars. → 2 column figure (both parts together or separate, depending on preference) Figure 8: Comparison of significance (non-significance, significant and very significant) of SF per polymer without taking the extent of supersaturation into account. → 1 column figure

Page 24 of 49

Table captions: Table 1: Classification of selected API based on their acidic and basic characteristics. Table 2: Log P and Log D values at pH = 1.6; 4.98; 5.0; 6.05; 6.5 and 7.18 for carbamazepine, cinnarizine, darunavir, diazepam, fenofibrate, griseofulvin, indomethacin, itraconazole, ketoconazole, naproxen, nifedipine and ritonavir predicted with MarvinSuite software (ChemAxon). Table 3: Supersaturation factors (SFs) in presence of biopolymers determined in 15 min – 120 min interval (See Eq. 1). Supersaturation factors in this table were calculated using solubility and supersaturation values both obtained in presence of the biopolymers. Table 4: Supersaturation factors (SFs) in presence of biopolymers determined in 15 min – 120 min interval. Supersaturation factors in this table were calculated using solubility data in absence of the biopolymer and supersaturation values obtained in presence of the biopolymer. Table 5: Influence of different Degree of Supersaturation (DS) on calculated Supersaturation factors (SF) of diazepam in aqueous media and naproxen in FaSSGF. Table 6: Summary on solid state, crystallinity and/or amorphicity, of XR(P)D-analysis of collected precipitates (See Appendix G for concomitant XR(P)D-analysis profiles). Table 7: Supersaturation factors (SFs) in presence of (semi-) synthetic polymers determined in 15 min – 120 min interval. Supersaturation factors in this table were calculated using solubility and supersaturation values both obtained in presence of the (semi-) synthetic polymer(s). Table 8: Supersaturation factors (SFs) in presence of (semi-) synthetic polymers determined in 15 min – 120 min interval. Supersaturation factors in this table were calculated using solubility in absence of the biopolymer and supersaturation values obtained in presence of the (semi-) synthetic polymer.

Page 25 of 49

References: [1] S. Stegemann, F. Leveiller, D. Franchi, H. de Jong and H. Lindén, Conference report: When poor solubility becomes an issue: From early stage to proof of concept. European Journal of Pharmaceutical Sciences, 31 (2007), 249-261. [2] C.A. Lipinski (Pfizer), Poor aqueous solubility – An industry wide problem in drug discovery. American Pharmaceutical Review, 5(3) (2002), 82-85. [3] A.M. Thayer and C&EN Houston, FINDING SOLUTIONS – Custom manufacturers take on DRUG SOLUBILITY ISSUES to help pharmaceutical firms move products through development. Chemical and Engineering News, 88(22) (2010), 13-18. [4] L.Z. Benet, The Role of BCS (Biopharmaceutics Classification System) and BDDCS (Biopharmaceutics Drug Disposition Classification System) in Drug Development. Journal of Pharmaceutical Sciences, 102(1) (2013), 34-42. [5] S. Kalepu and V. Nekkanti, Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharmaceutica Sinica B, 2015. 5(5) (2015): p. 442-453. [6] C. Leuner and J. Dressman, 2000. Improving drug solubility for oral delivery using solid dispersions. European Journal of Pharmaceutics and Biopharmaceutics, 50 (2000), 47-60. [7] G. Van den Mooter, The use of amorphous solid dispersions: A formulation strategy to overcome poor solubility and dissolution rate. Drug Discovery Today: Technologies, 9(2) (2012), 79-85. [8] Janssens and Van den Mooter, 2009 S. Janssens S. and G. Van den Mooter, Review: physical chemistry of solid dispersions. Journal of Pharmacy and Pharmacology, 61 (2009), 1571-1586. [9] T. Van Duong and G. Van den Mooter, REVIEW: The role of the carrier in the formulation of pharmaceutical solid dispersions. Part I: crystalline and semi-crystalline carriers. EXPERT OPINION ON DRUG DELIVERY (2016). [10] T. Van Duong and G. Van den Mooter, REVIEW: The role of the carrier in the formulation of pharmaceutical solid dispersions. Part II: amorphous carriers. EXPERT OPINION ON DRUG DELIVERY (2016). [11] C.L-N Vo, C. Park and B-J. Lee, Current trends and future perspectives of solid dispersions containing poorly watersoluble drugs. European Journal of Pharmaceutics and Bioprharmaceutics, 85 (2013), 799-813.

Page 26 of 49

[12] T. Vasconcelos, B. Sarmento and P. Costa, Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discovery Today, 12 (2007), 1068-1075. [13] Y. Lian, Amorphous pharmaceutical solids: preparation, characterization and stabilization. Advanced Drug Delivery Reviews, 48 (2001), 27-42. [14] B.C. Hancock and G. Zografi, Characteristics and significance of the amorphous state in pharmaceutical systems. Journal of Pharmaceutical Sciences, 86(1) (1997), 1-12. [15] D.Q.M. Craig, P.G. Royall, V.L. Kett and M.L. Hopton, The relevance of the amorphous state to pharmaceutical dosage forms: glassy drugs and freeze dried systems. International journal of pharmaceutics, 179 (1999), 179-207. [16] Y. Kawabata, K. Wada, M. Nakatani, S. Yamada and S. Onoue, Mini review: Formulation design for poorly water soluble drugs based on biopharmaceutics classification system: Basic approaches and practical applications. International Journal of Pharmaceutics, 420 (2011), 1-10. [17] N. Wyttenbach and M. Kuentz, Glass-forming ability of compounds in marketed amorphous drug products. European Journal of Pharmaceutics and Biopharmaceutics, 112 (2017), 204-208. [18] Y. Huang and W-G Dai, Fundamental aspects of solid dispersion technology for poorly soluble drugs. Acta Pharmaceutica Sinica B, 4 (2014), 18-25. [19] Y. He and C Ho, Review: Amorphous solid dispersions: utilization and challenges in drug discovery and development. Journal of Pharmaceutical Sciences, 104 (2015), 3237-3258. [20] T. Pas, B. Vergauwen and G. Van den Mooter, Exploring the feasibility of the use of biopolymers as a carrier in the formulation of amorphous solid dispersions – Part I: Gelatin. International Journal of Pharmaceutics, 535 (2018), 47-58. [21] S. Schmidt, D. Gonzales and H. Derendorf, Significance of protein binding in pharmacokinetics and pharmacodynamics. JOURNAL OF PHARMACEUTICAL SCIENCES, 99(3) (2010), 1107-1122. [22] F. Zhang, J. Xue, J. Shao and L. Jia, Compilation of 222 drugs’ plasma protein binding data and guidance for study designs. Drug Discovery Today, 17 (2012), 475-485. [23] T. Bohnert and L.-S. Gan, Plasma Protein Binding: From Discovery to Development. JOURNAL OF PHARMACEUTICAL SCIENCES, 102(9) 2013, 2953-2994. [24] I. Vroman and L. Tighzert, Biodegradable Polymers. Materials, 2 (2009), 307-344.

Page 27 of 49

[25] T. Imai, Y. Saito, H. Matsumoto, T. Satoh and M. Otagiri, Influence of egg albumin on dissolution of several drugs. International Journal of Pharmaceutics, 53 (1988), 7-12. [26] A. Bani-Jaber, I. Alshawabkeh, S. Abdullah, I. Hamdan, A. Ardakani, and M. Habash, In Vitro and In Vifo Evalution of Casein as a Drug Carrier for Enzymatically Triggered Dissolution Enhancement from Solid Dispersions. American Association of Pharmaceutical Scientists PharmSciTech, 1 (2016), 110. [27] H. Hsein, G. Garrait, M.A. Mumin, E. Beyssac and V. Hoffart, Atomization of denatured whey proteins as a novel and simple way to improve oral drug delivery system properties. International Journal of Biological Macromolecules, 105 (2017), 801-809. [28] M. Khoder, H. Abdelkader, A. ElShaer, A. Karam, M. Najlah and R.G. Alany, Efficient approach to enhance drug solubility by particle engineering of bovine serum albumin. International Journal of Pharmaceutics, 515 (2016), 740-748. [29] M. Khoder, H. Abdelkader, A. ElShaer, A. Karam, M. Najlah and R.G. Alany, The Use of Albumin Solid Dispersion to Enhance the Solubility of Unionisable Drugs. Pharmaceutical Development and Technology, (2017), 1-7. [30] R. Ojarinta, A.T. Heikkinen, E. Sievänen and R. Laitinen, Dissolution behavior of co-amorphous amino acid-indomethacin mixtures: The ability of amino acids to stabilize the supersaturated state of indomethacin. European Journal of Pharmaceutics and Biopharmaceutics, 112 (2017), 85-95. [31] K. Löbmann, H. Grohganz, R. Laitinen, C. Strachan and T. Rades, Amino acids as co-amorphous stabilizers for poorly water soluble drugs – Part 1: Preparation, stability and dissolution enhancement. European Journal of Pharmaceutics and Biopharmaceutics, 85 (2013), 873-881. [32] K. Löbmann, R. Laitinen, C. Strachan, T. Rades and H. Grohganz, Amino acids as co-amorphous stabilizers for poorly water soluble drugs – Part 2: Molecular interactions. European Journal of Pharmaceutics and Biopharmaceutics, 85 (2013), 882-888. [33] G. Kasten, H. Grohganz, T. Rades and K. Löbmann, Development of a screening method for coamorphous formulations of drugs and amino acids. European Journal of Pharmaceutical Sciences, 95 (2016), 28-35. [34] K.M. Ropella, Introduction to Statistics for Biomedical Engineers. E-book, Morgan & Claypool, 2007. (DOI: DOI: 10.2200/S00095ED1V01Y200708BME014) [35] C. Heamann and M.S. Shalabh, Introduction to Statistics and Data Analysis. Switzerland, Springer Nature, 2016. (DOI 10.1007/978-3-319-46162-5) Page 28 of 49

[36] D.M. Lane, D. Scott, M. Hebl, R. Guerra, D. Osherson and H. Zimmer, Introduction to Statistics. (online edition) [37] Y. Ni, R. Zhu and S. Kokot, Compertitive binding of small molecules with biopolymers: a fluorescence spectroscopy and chemometrics study of the interachtion of aspirin and ibuprofen with BSA. Analyst, 136 (2011), 4794-4801.

Page 29 of 49

Figure 1:

Itraconazole Carbamazepine

Cinnarizine

Diazepam

Indomethacin

Darunavir Nifedipine

Ritonavir

Fenofibrate

Ketonconazole Griseofulvin

Naproxen

Page 30 of 49

Figure 2:

a) Carbamazepine

b) Cinnarizine m + 1% BSA m + 1% 50PS Medium

0,192 0,170 0,142

0,423

FaSSGF

0,187 0,160 0,146

0,005 0,001 3,105E-04

FaSSIF

0,204

H2O

0,005 0,008 5,766E-04

H2O

0,150 0,189

0,0

0,1

0,2

0,3

0,00

Concentration (mg/mL)

2,255 2,093 2,431

H2O 0,00

0,106

0,000

0,079 0,046 0,046

m + 1% BSA m + 1% 50PS Medium

0,002 6,823E-05 5,237E-05

0,035 0,032 0,032 0,011

FaSSIF

m + 1% BSA m + 1% 50PS Medium

0,98

1,96

Concentration (mg/mL)

2,94

H2O 0,000

0,178

5,016E-04

0,005 7,624E-04 4,527E-04

0,027

FaSSGF

0,020 0,014 0,045

FeSSIF

0,039 0,036 0,026 0,025 0,022

FaSSIF

0,022

H2O 0,014

0,267

Concentration (mg/mL)

0,009

0,070 0,041 0,023

0,089

f) Griseofulvin

FeSSIF

Medium

Medium

FaSSIF

0,252 0,207 0,204

FaSSGF

m + 1% BSA m + 1% 50PS Medium 0,177

0,51

e) Fenofibrate

FaSSGF

H2O

Concentration (mg/mL)

d) Diazepam

FeSSIF

0,34

0,117 0,100 0,127

FaSSIF

0,139

m + 1% BSA m + 1% 50PS Medium

0,17

0,173 0,162 0,158

FeSSIF

Medium

Medium

Medium

0,132 0,122 0,132

FeSSIF

0,223 0,208

FaSSIF

0,214 0,209

0,024 0,296

FeSSIF

0,248

FaSSGF

0,263

Medium

FaSSGF

c) Darunavir

0,028

Concentration (mg/mL)

0,042

m + 1% BSA m + 1% 50PS Medium

0,014 0,013

0,000

0,016

0,032

0,048

Concentration (mg/mL)

Page 31 of 49

g) Indomethacin

h) Itraconazole m + 1% BSA m + 1% 50PS Medium

0,007

FaSSGF 0,001 0,002

0,002 0,002

FaSSGF

0,712 0,300 0,290

0,001

0,56

0,84

0,0000

0,0017

Concentration (mg/mL)

m + 1% BSA m + 1% 50PS Medium

0,030 0,018 0,018 0,356 0,220 0,209

1,574

0,0

FaSSGF

0,027 0,021 0,021

H2O

0,024 0,078 0,046

m + 1% BSA m + 1% 50PS Medium

0,0

4,2

1,337 1,341 0,076 0,065 0,073

m + 1% BSA m + 1% 50PS Medium 0,064 0,060 0,052

0,032 0,029 0,033

FaSSGF 0,010 0,009

FeSSIF

0,011 0,013

0,004 0,008

FaSSIF

0,002 0,003

H2O

0,002

0,023

H2O 0,6

1,2

Concentration (mg/mL)

1,8

0,000

0,010

0,010 0,010

m + 1% BSA m + 1% 50PS Medium

0,007

0,027

12,6

Concentration (mg/mL)

0,025

FaSSIF

8,4

l) Ritonavir

0,015 0,009 0,011

FeSSIF

FaSSIF

H2O

0,0051

k) Nifedipine

Medium

Medium

FeSSIF

FaSSIF

Concentration (mg/mL)

j) Naproxen FaSSGF

0,0034

Medium

0,28

0,001 7,718E-04

2,397 0,644

0,002

H2O

0,013 0,021

0,00

m + 1% BSA m + 1% 50PS Medium

0,001 9,243E-04 0,002

FaSSIF

0,312

H2O

0,092

FeSSIF

0,003

Medium

Medium

Medium

9,315E-04

FeSSIF

0,128 0,122

FaSSIF

10,287 9,165 8,216

FaSSGF

0,001

0,209

FeSSIF

i) Ketoconazole

0,054

Concentration (mg/mL)

0,081

0,000

0,014

0,028

Concentration (mg/mL)

Page 32 of 49

0,042

Figure 3:

a) Carbamazepine

b) Cinnarizine

1,0

Charge

c) Darunavir

0,5

Cinnarizine Charge

Charge

Carbamazepine Charge

Charge

0,5

1,5

0,0

1,0

Charge

2,0

1,0

Darunavir

0,0

-0,5 -0,5

0,5

-1,0

0,0

-1,0 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

0

1

2

3

4

5

6

7

8

9

0

10 11 12 13 14

1

2

3

4

5

6

d) Diazepam

e) Fenofibrate

8

9

10

11

12

13

14

f) Griseofulvin

1,0

Charge

1,0

7

pH

pH

pH

1,0

Charge

0,5

Charge

0,5 Griseofulvin

Fenofibrate 0,5

Charge

Charge

Charge

Diazepam 0,0

-0,5

0,0

-0,5

-1,0 0

1

2

3

4

5

6

7

pH

8

9

10 11 12 13 14

0,0

-1,0 0

1

2

3

4

5

6

7

pH

8

9

10 11 12 13 14

0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

pH

Page 33 of 49

g) Indomethacin

h) Itraconazole Charge

0,0

i) Ketoconazole Charge

2,0

Charge

2,0 1,8

1,5

1,6

Itraconazole

Ketoconazole

1,4

Indomethacin -0,5

Charge

Charge

Charge

1,2 1,0

1,0 0,8 0,6

0,5

0,4 0,2

-1,0

0,0 0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

0,0 0

1

2

3

4

5

6

pH

7

8

9

10 11 12 13 14

0

1

2

3

4

5

6

pH

j) Naproxen

k) Nifedipine Naproxen

8

9

10 11 12 13 14

l) Ritonavir

1,0

Charge

0,0

7

pH

2

Charge

0,5

Charge

1

Ritonavir

-0,5

Charge

Charge

Charge

Nifedipine 0,0

0

-0,5 -1 -1,0

-1,0 0

1

2

3

4

5

6

7

pH

8

9

10 11 12 13 14

0

1

2

3

4

5

6

7

pH

8

9

10 11 12 13 14

0

1

2

3

4

5

6

7

8

9

10 11 12 13 14

pH

Page 34 of 49

Figure 4: b) Concentration

Concentration

a) 15 min – 120 min time range

Legend: Drug solubility in medium Drug solubility in medium + polymer Drug supersaturation in medium + polymer

A

AUC15mi n-120mi n supersaturation

A

B

AUC15mi n-120mi n solubility

B

= SF

15 min – 120 min time range

A

B Time

Time

Page 35 of 49

Figure 5: H2O - solu H2O 1% BSA - solu H2O 1% BSA - sup (1%) H2O 1% BSA - sup (2%)

6.0 5.5

Concentration (mg/mL)

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

10

20

30

40

50

60

70

80

90

100

110

120

Time (min)

Page 36 of 49

Figure 6: FeSSIF - solu FeSSIF 1% BSA - solu FeSSIF 1% BSA - sup (non-homogenized) FeSSIF 1% BSA - sup (homogenized)

4.0

Concentration (mg/mL)

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0

10

20

30

40

50

60

70

80

90

100

110

120

Time (min)

Page 37 of 49

Figure 7:

a) Carbamazepine

b) Cinnarizine

c) Darunavir H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,0013

0,2260

0,2333

0,0030

0,2295

0,1705

H2O

H2O

H2O

2,1924E-04

0,3252

0,1886

0,00

0,12

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,24

0,36

0,1767

5,7661E-04

0,000

0,002

0,004

0,006

Solubility (mg/mL)

Solubility (mg/mL)

d) Diazepam

0,1290

0,0294

0,0214

0,0014

H2O

H2O

0,033

0,066

Solubility (mg/mL)

0,0203

7,5893E-04

0,0579

0,000

0,099

0,33

f) Griseofulvin

0,0867

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,22

0,0012

0,0879

0,0226

0,11

Solubility (mg/mL)

e) Fenofibrate

H2O

0,00

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

4,5275E-04

0,0000

0,0007

0,0014

Solubility (mg/mL)

0,0021

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,0129

0,000

0,012

0,024

Solubility (mg/mL)

Page 38 of 49

0,036

g) Indomethacin

h) Itraconazole H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,014

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

3,37E-04

0,017

0,113

H2O

H2O

0,031

6,06E-04

0,021

0,017

0,067

3,37E-04

H2O

0,000

i) Ketoconazole

0,016

7,72E-04

0,034

0,051

0,0000

0,0005

Solubility (mg/mL)

0,0010

0,0015

0,000

0,041 0,082 Solubility (mg/mL)

Solubility (mg/mL)

j) Naproxen

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,046

k) Nifedipine

0,123

l) Ritonavir

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,0053 0,0302

0,0952

0,0035 0,0234

0,0649

H2O

H2O

H2O

0,0029 0,0658

0,0155

H2O 1% PVPVA solu H2O 1% PVP solu H2O 1% HPMC solu H2O solu

0,0096 0,0734

0,000

0,034

0,068

Solubility (mg/mL)

0,102

0,000

0,012

0,024

Solubility (mg/mL)

0,036

0,0073

0,000

0,004

0,008

Solubility (mg/mL)

Page 39 of 49

0,012

Figure 8:

very significant (p<0.01) significant (p<0.05) non-significant (p>0.05)

No polymer

gelatin 50PS

BSA

HPMC

PVP

PVPVA

comparison of significance of SF / polymer

Page 40 of 49

Table 1 Weak acids Indomethacin Naproxen

Weak bases Cinnarizine Diazepam Itraconazole Ketoconazole

Neutral drugs Carbamazepine Fenofibrate Griseofulvin Nifedipine

Amphoteric drugs Darunavir Ritonavir

Page 41 of 49

Table 2 API

Log P

Carbamazepine Cinnarizine Darunavir Diazepam Fenofibrate Griseofulvin Indomethacin Itraconazole Ketoconazole Naproxen Nifedipine Ritonavir

2.77 6.24 2.82 3.08 5.28 2.17 3.53 7.31 4.19 2.99 1.82 5.22

pH = 1.6 FaSSGF 2.77 1.56 1.98 1.74 5.28 2.17 3.53 4.47 1.32 2.98 1.82 4.20

pH = 4.98 1% gelatin 50PS in purified H2O 2.77 2.74 2.82 3.07 5.28 2.17 2.33 7.28 3.58 2.13 1.82 5.22

Log D pH = 5.0 pH = 6.05 FeSSIF Purified H2O 2.77 2.77 2.74 2.81 2.82 2.82 3.07 3.08 5.28 5.28 2.17 2.17 2.31 1.30 7.28 7.31 3.58 3.77 2.11 1.13 1.82 1.82 5.22 5.22

pH = 6.5 FaSSIF 2.77 2.90 2.82 3.08 5.28 2.17 0.89 7.31 3.91 0.70 1.82 5.22

pH = 7.18 1% BSA in purified H2O 2.77 3.24 2.82 3.08 5.28 2.17 0.38 7.31 4.09 0.11 1.82 5.22

Page 42 of 49

Table 3 Aqueous media (pH, see below) Drug Carbamazepine Cinnarizine Ritonavir Darunavir Diazepam Griseofulvin Indomethacin Nifedipine Naproxen Ketoconazole Fenofibrate Itraconazole

1% gelatin 50PS (pH 4.98 ± 0.01)

1% BSA (pH 7.18 ± 0.01)

FaSSIF (pH 6.50 ± 0.01) 1% gelatin 50PS

FeSSIF (pH 5.00 ± 0.01)

1% BSA

1% gelatin 50PS

1% BSA

FaSSGF (pH 1.60 ± 0.01) 1% gelatin 50PS

1% BSA 1.38

1.18

1.11

1.82

1.31

1.51

1.03

1.28

11.06

2.80

17.49

11.58

8.55

7.70

11.98

9.08

12.50

5.35

15.28

9.39

10.90

10.98

14.31

13.53

2.21

2.27

3.70

3.16

3.07

1.49

2.64

2.52

8.71

7.82

2.58

7.95

6.90

6.35

1.49

1.90

2.76

2.22

0.40

1.10

1.64

2.68

1.04

1.01

11.47

7.65

9.82

1.78

7.65

9.03

11.30

7.55

1.71

1.60

1.00

1.91

1.13

1.49

1.61

1.61

1.80

4.83

1.17

1.14

3.54

2.31

1.25

1.57

2.51

4.56

4.27

4.64

/

/

/

/

1.00

2.96

10.19

0.99

0.89

1.65

3.23

1.35

2.06

1.03

7.89

7.43

3.36

12.16

5.63

20.51

Legend: = very significant (p ≤ 0.01) (= significantly different on CI 95% and CI99%) = significant (p ≤ 0.05) (= significantly different on CI95%) = Non-significant (p > 0.05) (= non-significantly different on CI 95%) Supersaturation Factors (SF) calculated as: ss(m + 1%) / sol(m + 1%) (with ss = supersaturation and sol = solubility and m = medium)

Page 43 of 49

Table 4 Aqueous media (pH, see below) Drug Carbamazepine Cinnarizine Ritonavir Darunavir Diazepam Griseofulvin Indomethacin Nifedipine Naproxen Ketoconazole Fenofibrate Itraconazole

1% gelatin 50PS (pH 4.98 ± 0.01)

1% BSA (pH 7.18 ± 0.01)

FaSSIF (pH 6.50 ± 0.01) 1% gelatin 50PS

FeSSIF (pH 5.00 ± 0.01)

1% BSA

1% gelatin 50PS

1% BSA

FaSSGF (pH 1.60 ± 0.01) 1% gelatin 50PS

1% BSA

0.94

1.21

1.99

1.67

1.61

1.46

1.53

1.86

157.49

25.44

84.11

197.85

7.90

7.71

132.33

161.21

4.05

7.61

11.93

26.07

23.78

24.74

12.50

13.15

1.33

1.79

2.90

2.91

3.13

1.57

2.69

2.99

15.72

24.11

2.60

13.67

7.00

7.82

1.28

1.76

3.04

3.72

0.48

1.34

1.76

3.30

1.48

1.94

7.27

113.92

10.13

4.37

8.03

15.53

7.94

25.46

1.72

3.85

0.83

3.66

1.29

1.82

1.38

2.20

1.59

5.02

1.16

1.34

4.23

2.39

4.32

6.06

3.72 /

3.94 /

1.25 /

2.56 /

1.68

31.26

0.57

1.20

0.90

1.79

4.21

42.70

3.02

2.87

3.79

5.34

7.33

8.27

10.80

39.08

Supersaturation Factors (SF) calculated as: ss(m + 1%) / sol(m) (with ss = supersaturation and sol = solubility and m = medium)

Page 44 of 49

Table 5 Aqueous media FaSSGF (pH 1.6) Drug DS 1% gelatin 1% BSA 1% gelatin 1% BSA Diazepam DS 20 6.65 6.22 DS 10 8.71 7.82 Naproxen DS 10 0.98 1.58 DS 5 1.28 1.57

Page 45 of 49

Table 6 H2O Drug Carbamazepine Cinnarizine Darunavir Diazepam Fenofibrate Griseofulvin Indomethacin Itraconazole Ketoconazole Naproxen Nifedipine Ritonavir

FaSSGF

FaSSIF

FeSSIF

No polymer

1% Gelatin 50PS

1% BSA

1% HPMC

1% PVP

1% PVPVA

1% Gelatin 50PS

1% BSA

1% Gelatin 50PS

1% BSA

1% Gelatin 50PS

1% BSA

C* A A C A C C* A A C C A

C* C A A C C A A A* C C A

C* A* A A C C C* A A A C A

C* A A A A C A A A C C A

C* A A C A C A A C C C A

C* A A A A C A A C C C A

C* C A C A C A A A C C A*

C* C A C C C A* A* A C C A*

C* A A C C C C* A / C C A*

C* C A C C C C* A / C C A*

C* C A C C C C* A* / C C A*

C* C A C C C C* A* / C C A*

Legend: A = X-ray amorphous A* = X-ray amorphous, although reflections seemed to be present not indistinguishable from noise C = X-ray crystalline, same crystalline form as starting material C* = X-ray crystalline, different crystalline form than starting material, other polymorph, partially other polymorph? For XR(P)D-profiles, readers are referred to appendix G

Page 46 of 49

Table 7 Aqueous media Drug Carbamazepine Cinnarizine Ritonavir Darunavir Diazepam Griseofulvin Indomethacin Nifedipine Naproxen Ketoconazole Fenofibrate Itraconazole

No polymer (pH = 6.05 ± 0.035)

1% HPMC (pH = 4.74 ± 0.01)

1% PVP (pH = 4.00 ± 0.01)

1% PVPVA (pH = 4.29 ± 0.01)

1.43 3.83 4.45 2.09 9.62 1.08 0.80 1.36 0.91 1.17 1.53 0.59

1.13 17.67 13.19 3.15 7.45 2.23 3.61 7.49 0.92 4.54 1.26 1.71

1.55 18.15 11.84 2.82 5.24 1.09 4.08 0.98 1.21 1.99 0.54 2.18

1.54 18.35 8.80 2.20 5.63 2.74 5.37 9.74 0.97 1.81 0.83 2.72

Legend: = very significant (p ≤ 0.01) (= significantly different on CI95% and CI99%) = significant (p ≤ 0.05) (= significantly different on CI 95%) = Non-significant (p > 0.05) (= non-significantly different on CI95%) Supersaturation Factors (SF) calculated as: ss(m + 1%) / sol(m + 1%) (with ss = supersaturation and sol = solubility and m = medium)

Page 47 of 49

Table 8 Aqueous media Drug Carbamazepine Cinnarizine Ritonavir Darunavir Diazepam Griseofulvin Indomethacin Nifedipine Naproxen Ketoconazole Fenofibrate Itraconazole

No polymer (pH = 6.05 ± 0.035)

1% HPMC (pH = 4.74 ± 0.01)

1% PVP (pH = 4.00 ± 0.01)

1% PVPVA (pH = 4.29 ± 0.01)

1.43 3.83 4.45 2.09 9.62 1.08 0.80 1.36 0.91 1.17 1.53 0.59

1.96 6.72 5.21 2.30 19.10 3.50 5.38 12.05 0.84 1.59 2.12 1.34

1.88 93.46 5.66 2.72 20.11 1.82 3.40 2.37 1.07 4.84 1.69 0.95

1.85 39.97 6.35 2.91 21.90 6.25 3.53 30.51 1.26 2.60 2.22 1.19

Supersaturation Factors (SF) calculated as: ss(m + 1%) / sol(m) (with ss = supersaturation and sol = solubility and m = medium)

Page 48 of 49

Graphical abstract

Solubility by shake-flask Determination of equilibrium solubility of drug in medium (with and without polymer)

Supersaturation by solvent-shift Addition of concentrated drug solution (organic solvent)

Concentration

Drug Supersaturation in the presence of polymer

Polymer induced supersaturation

Drug solubility in the presence of polymer

Drug solubility in medium

Time Polymer containing medium

* Icons from flaticon.com

Page 49 of 49