# Bosentan monohydrate and sildenafil base as two companions in enabling formulations

<figure><img src="image_1.png" /></figure>

Dominik Strojewski<sup>a, b</sup>, Sebastian Lalik<sup>c</sup>, Florence Danède<sup>d</sup>, Natalia Górska<sup>e</sup>, Aleksandra Deptuch<sup>f</sup>, Monika Marzec<sup>c</sup>, Jean-François Willart<sup>d</sup>, Anna Krupa<sup>a, d, *</sup>

## ARTICLE INFO

### Keywords:

Combination drugs

Mixture drug products

Amorphous solid dispersions

Molecular alloys

Supersaturated systems

Poorly soluble drugs

Mechanical activation

High energy ball milling

## ABSTRACT

**Hypothesis:** Sildenafil base and bosentan monohydrate are co-administered in a chronic therapy of pulmonary arterial hypertension (PAH). Both drugs are poorly soluble in water, and their bioavailability is limited to ca. 50 %. Since bosentan is a weak acid, whereas sildenafil is a weak base, we assumed that their co-amorphization could: (i) improve their solubility in the gastrointestinal fluids, (ii) enable to reach supersaturation and (iii) ensure stabilization of supersaturated solutions. If successful, this could accelerate the development of new fixed-dose combination drugs. Experiments: The co-amorphous formulations were prepared using high energy ball milling. Their solid state properties were assessed using XRD, DSC, FT-MIR, and dielectric spectroscopy. Particle size distribution and surface wetting were also analyzed. Polarizing optical microscopy and scanning electron microscopy were applied to assess the microstructure of these powders. A new HPLC-DAD method was developed for a simultaneous quantification of both drugs. Findings: It was shown that binary formulations in which bosentan was molecularly dispersed in sildenafil base (Tg = 64-78 °C) could be manufactured in the high energy ball milling process. When the sildenafil load was below 50 wt. %, the formulations showed the greatest thermal stability and formed long-lasting bosentan supersaturation in PBS.

## 1. Introduction

Pulmonary arterial hypertension (PAH) is a progressive, leading to death cardio-pulmonary disease (Tan et al., 2022). It is characterized by elevated blood pressure in the pulmonary arteries, e.g. > 20 mmHg, resulting in a pulmonary vasculature that leads to right heart failure. PAH therapy is based on three separate signaling pathways, namely: (i) the endothelin pathway, (ii) the prostacyclin pathway and (iii) the NO/cGMP pathway (Tamura and Kimura, 2015).

Endothelin 1 causes constriction of pulmonary vessels and stimulates proliferation of smooth muscle cells. This action leads to an increase in pulmonary blood pressure and worsening of the disease. Thus, the administration of endothelin receptor antagonists helps relieve the symptoms of PAH and slows the progression of the disease. Bosentan

monohydrate is a sulfonamide derivative and a nonselective, nonpeptide antagonist of endothelin receptors. The presence of a sulfonamide group in its structure is responsible for binding to these receptors and acidic properties. As a consequence, the drug is poorly soluble at gastric pH (ca. 2 µg mL⁻¹), but when the pH is 6.80 or higher its solubility increases to more than 40 µg mL⁻¹. Despite this fact, the oral bioavailability of bosentan is limited to 50 %. In 2001, Actelion, Ltd. launched bosentan hydrate for the therapy of PAH in the US under a brand name Tracleer. A year later, the drug was introduced to the EU market by Janssen-Cilag International.

The results of the *meta*-analysis (Kuang et al., 2021) confirm that long-term oral administration of bosentan is well tolerated and effective. Furthermore, new clinical indications for bosentan have also been discovered throughout recent years, not only stimulating the

<footnote><sup>a</sup> Jagiellonian University, Medical College, Faculty of Pharmacy, Department of Pharmaceutical Technology and Biopharmaceutics, PL-30688 Cracow, Poland</footnote>

<footnote><sup>b</sup> Doctoral School of Medical and Health Sciences, Jagiellonian University in Cracow, PL-31530, Poland</footnote>

<footnote><sup>c</sup> Jagiellonian University, Institute of Physics, PL-30348 Cracow, Poland</footnote>

<footnote><sup>d</sup> University of Lille, CNRS, INRAE, Centrale Lille, UMR 8207, UMET – Unité Matériaux et Transformations, F-59000 Lille, France</footnote>

<footnote><sup>e</sup> Jagiellonian University, Faculty of Chemistry, PL-30387 Cracow, Poland</footnote>

<footnote><sup>f</sup> Institute of Nuclear Physics Polish Academy of Sciences, PL-31342 Cracow, Poland</footnote>

<footnote>* Corresponding author at: Jagiellonian University, Medical College, Faculty of Pharmacy, Department of Pharmaceutical Technology and Biopharmaceutics, 9 Medyczna Street, 30-688 Cracow, Poland.</footnote>

<footnote>E-mail address: a.krupa@uj.edu.pl (A. Krupa).</footnote>

<footnote>https://doi.org/10.1016/j.ijpharm.2024.124312</footnote>

<footnote>Received 26 January 2024; Received in revised form 29 May 2024; Accepted 4 June 2024</footnote>

<footnote>Available online 13 June 2024</footnote>

<footnote>0378-5173/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>
<table><caption>**Table 1**
The properties of bosentan monohydrate and sildenafil base.</caption><thead><tr><th></th><th>Bosentan monohydrate</th><th>Sildenafil base</th></tr></thead><tbody><tr><td>Molecular structure</td><td><img src=" bosentan_structure.png" alt="Chemical structure of bosentan monohydrate"></td><td><img src=" sildenafil_structure.png" alt="Chemical structure of sildenafil base"></td></tr><tr><td>Class of organic compounds</td><td>Bipyrimidines &amp; Oligopyrimidines</td><td>Benzenesulfonamides</td></tr><tr><td>Formula</td><td>C<sub>27</sub>H<sub>29</sub>N<sub>5</sub>O<sub>6</sub>S·H<sub>2</sub>O</td><td>C<sub>22</sub>H<sub>30</sub>N<sub>6</sub>O<sub>4</sub>S</td></tr><tr><td>M<sub>w</sub> (g/mol)</td><td>569.6</td><td>474.6</td></tr><tr><td>Crystal size (mm<sup>3</sup>)</td><td>0.79 x 0.43 x 0.22</td><td>0.40 x 0.30 x 0.05</td></tr><tr><td>Crystal system</td><td>Monoclinic</td><td></td></tr><tr><td>Space group</td><td>P2<sub>1</sub>/c</td><td></td></tr><tr><td>Bonds stabilizing structure</td><td>O—H···O, O—H···N &amp;<br/>N—H···O hydrogen bonds with a single water molecule, &amp;<br/>weak O—H···N intermolecular interactions between the<br/>hydroxy group and one of the pyrimidine rings producing an<br/>two-dimensional supramolecular array in the <i>bc</i> plan</td><td>One intramolecular hydrogen bond,<br/>Crystal structure stabilized by van der Waals forces</td></tr><tr><td>pKa</td><td>5.46</td><td>5.99</td></tr><tr><td>logP</td><td>3.70</td><td>1.90</td></tr><tr><td>Bioavailability (%)</td><td>50</td><td>41</td></tr><tr><td>Drug classification</td><td>Dual endothelin receptor antagonist</td><td>Selective inhibitor of cGMP-specific phosphodiesterase type 5 (PDE-5)</td></tr><tr><td>Clinical indications</td><td>Pulmonary arterial hypertension,<br/>Systemic sclerosis (Scleroderma),<br/>Eisenmenger's syndrome,<br/>Ocular inflammation,<br/>Ocular bacterial infection</td><td>Erectile dysfunction,<br/>Pulmonary arterial hypertension</td></tr><tr><td>Derivatives</td><td>Alkali metal salts<br/>Ammonium salt</td><td>Sildenafil citrate monohydrate<br/>Sildenafil saccharinate</td></tr><tr><td>Drug doses (mg)</td><td>32, 62.5; 125</td><td>25–100</td></tr><tr><td>Brand names</td><td>Stayveer, Tracleer</td><td>Revatio, Viagra, Vizarsin</td></tr><tr><td>Available dosage forms</td><td>Tablets,<br/>Film-coated tablets,<br/>Dispersible tablets,<br/>Ophtalmic suspension</td><td>Oral suspension,<br/>Coated tablets,<br/>Chewable tablets</td></tr><tr><td>Group of patients</td><td>Children &amp; Adults</td><td></td></tr><tr><td>Combination drugs/Mixture products</td><td>Ophtalmic suspension Ocubrax: Bosentan 3 mg + Dexamethasone 1 mg</td><td>Tablets Dapoksel/Dapoxil: Sildenafil citrate 50 mg + Dapoxetine hydrochloride 30 mg</td></tr></tbody></table>

development of various brand and generic products (Table 1), but also encouraging modifications of its chemical structure. Among them, there are alkali metal or ammonium salts (Singh et al., 2012) of higher solubility than bosentan hydrate.

In turn, phosphodiesterase type 5 inhibitors, e.g. sildenafil, are used in the therapy of PAH according to the NO/cGMP pathway. Under physiological conditions, endogenous nitric oxide (NO), which is produced in endothelial cells, induces the synthesis of cyclic guanosine monophosphate (cGMP) in smooth muscles. Phosphodiesterase type 5 (PDE5) degrades cGMP, inhibiting its action. When sildenafil is administered, cGMP is not degraded by PDE5, and as a result its concentration increases together with NO, leading to a reduction in blood pressure in pulmonary vessels. In addition to vasodilation, nitric oxide also shows an antiproliferative effect on smooth muscle cells (Tamura and Kimura, 2015). From a chemical point of view, sildenafil is a derivative of 7H-pyrazolo[4,3-d]pyrimidin-7-one. The presence of the piperazine ring in its structure gives it basic character. A poorly soluble in water sildenafil base (53.90 ± 9.92 µg mL⁻¹) is capable to form salts with anions, and therefore; its equilibrium solubility increases significantly at gastric pH to reach the value of 26.78 ± 3.49 mg mL⁻¹, but then, it rapidly decreases to 28.61 ± 12.88 µg mL⁻¹ at pH = 6.80. Therefore, sildenafil citrate is used in therapy. However, its oral bioavailability is still relatively low, namely around 41 %. Despite this fact, sildenafil effectively reduces PAH symptoms, prolongs the time to clinical worsening, and improves the quality of life (Bhogal et al., 2019).

In 2016, the results of a *meta*-analysis comparing the efficacy of PAH monotherapy with combination therapy were published (Lajoie et al., 2016). They showed a reduced risk of clinical worsening when combination therapy with endothelin antagonists and PDE5 inhibitors was applied compared to the monotherapy. These findings are consistent with the conclusions drawn on a basis of the recent *meta*-analysis of randomized clinical trials that compares the efficacy of the monotherapy and combination therapy (Tan et al., 2022). They revealed that the latter approach improved exercise capacity and functional status of patients.

Taking into account the potential clinical advantages of the concomitant use of bosentan monohydrate and sildenafil base for the management of PAH together with their limited solubility and weak acid-weak base properties, it was assumed that the coprocessing of these two compounds could be a way to enhance their overall performance.

This assumption is consistent with the growing importance of fixed-dose combination drugs in the pharmacotherapy of arterial hypertension that has been observed in recent years. Clinical data confirm that they ensure better disease control and better compliance in chronic treatments compared to single drug preparations (Du et al., 2018). Moreover, the mixture products are being adapted also for other routes of drug administration than oral, for example, pulmonary delivery (Banat et al., 2023). Thus, the implementation of this strategy for the treatment of PAH can also be worth investigating, especially when combining drugs of well-established position in the therapy.

From a technological perspective, there are numerous examples of
successful combinations of poorly soluble drugs, which show therapeutic synergy. Among them, there are co-amorphous drug-drug systems loaded with atenolol and hydrochlorothiazide prepared by cryomilling (Moinuddin et al., 2017), or a combination of ponazuril and toltrazuril (Li et al., 2022). Since the molecular miscibility of the compounds is crucial for the stability of the co-amorphous formulation, ponazuril, an active metabolite of toltrazuril, was proposed to stabilize the amorphous form of this drug (Li et al., 2022). The similarity in the chemical structure of both drugs ensured their high miscibility and, in consequence, stable co-amorphous systems were obtained. Apart from the structural similarity, glass forming ability is one of the key features when selecting a proper solid dispersion coformer (Newman et al., 2018; Yarlagadda et al., 2021).

Recent studies provided evidence that amorphization of bosentan alone (Krupa et al., 2022; Minecka et al., 2022) or in combination with hydrophilic polymers, e.g. copovidone, enabled to considerably enhance dissolution rate, and to reach a long-lasting supersaturation (Krupa et al., 2023). Thermal treatment (melt quenching), mechanical activation of drug particles under high-energy ball milling, or solvent evaporation in nano spray drying ensured quick amorphization of bosentan. Importantly, its amorphous form was very stable in the solid state and did not crystallize upon heating (Krupa et al., 2023, 2022). The vitrification of sildenafil and the stability of its glass have also been studied in detail (Kolodziejczyk et al., 2014, 2013). In this case, the susceptibility to crystallization was highly dependent on the temperature. The sildenafil glass was stable for at least 6 months under ambient conditions (25 °C), but after heating above the glass transition temperature (Tg = 56 °C) and below the melting point (Tm ≈ 187 °C), cold crystallization occurred (Kolodziejczyk et al., 2013). This process relied on the diffusion of molecules that led to instantaneous and sporadic nucleation, and then 3D crystallites formed (Kolodziejczyk et al., 2014). However, the right choice of the polymer could prevent crystallization (Knapik-Kowalczuk et al., 2020).

In turn, the co-milling of bosentan monohydrate (weak acid) and sildenafil base (weak base) has not been studied before to the best of our knowledge. Therefore, the objective of the present study is to evaluate the impact that the mechanical activation of such binary systems has on the physical properties of these drugs in the solid state and, then, their ability to form supersaturated solutions in the gastrointestinal tract (GIT). Our attention is focused on the assessment of whether, under co-milling (i) co-amorphous drug-drug molecular alloys could be formed; (ii) to evaluate the strength of interactions between bosentan and sildenafil; (iii) to predict their stability, and to finally assess (iv) if they are able to create long-lasting supersaturation under physiological conditions.

The interactions between these drugs were investigated using various complementary analytical tools, e.g. scanning electron microscopy (SEM) and polarizing optical microscopy (POM), X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), and frequency domain dielectric spectroscopy (FDDS). The shifts of chemical groups within the structure of these compounds induced by their mechanical activation were analyzed using Fourier transform middle-infrared absorption spectroscopy (FT-MIR). Contact angle measurements were performed to assess the impact of amorphization on the wettability of their surface. The simultaneous release of sildenafil base and bosentan from binary formulations in the gastric or intestinal environment was evaluated in a modified paddle apparatus with a set of small-volume vessels using a high-performance liquid chromatography with diode array detection (HPLC-DAD).

## 2. Materials and methods

Bosentan monohydrate (BOS) was purchased from Biolim (Gdynia, Poland), while sildenafil base (SIL) was kindly donated by Polpharma S. A. (Starogard Gdański, Poland).

Analytical grade 36 % (v/v) hydrochloric acid was obtained from

<table><caption>**Table 2**
The composition of high energy ball milled samples.</caption><thead><tr><th>Sample name</th><th>BMO</th><th>BMO.2</th><th>BMO.5</th><th>BMO.8</th><th>BM1.0</th></tr></thead><tbody><tr><td>Bosentan monohydrate (g)</td><td>0.0</td><td>0.2</td><td>0.5</td><td>0.8</td><td>1.0</td></tr><tr><td>Sildenafil base (g)</td><td>1.0</td><td>0.8</td><td>0.5</td><td>0.2</td><td>0.0</td></tr></tbody></table>

Merck (Warsaw, Poland). Sodium chloride, sodium hydroxide, sodium phosphate monobasic anhydrous, potassium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland). All of these reagents were of analytical grade. Chromatography grade acetonitrile (ACN) and methanol (MeOH) were purchased from Witko (Łódź, Poland), while triethanolamine (TEA) and 85 % (v/v) orthophosphoric acid (PA) were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany) and VWR International (Gdańsk, Poland) respectively. 3F powder was purchased from Biorelevant (London, United Kingdom). A Milli-Q Elix Essential water purification system of Millipore Corporation (Merck, Warsaw, Poland) was used to prepare household chromatographic grade water.

### 2.1. Preparation of solid dispersions

High energy ball milling (BM) was carried out at room temperature (RT) using a Pulverisette 7, Fritsch (Idar-Oberstein, Germany) planetary ball mill. Binary mixtures (1.0 g) composed of BOS and SIL were placed in 45 mL milling jars made of zirconium oxide together with seven milling balls (Ø = 15 mm) of the same material, resulting in a ball-to-sample weight ratio of 75:1. The weight ratio of the drugs was investigated at three levels that corresponded to the following weight fractions of BOS 0.2; 0.5 and 0.8. Information on the weight fraction of BOS in the sample was also indicated by a number in the sample name (Table 2). The rotational speed of the solar disc was set at 400 rpm. The milling was performed for 240 min, but the milling periods of 20 min were alternated with pause periods of 10 min to avoid overheating of the samples. The milled samples were sieved (Ø = 180 µm) before analyses. For comparison, BOS and SIL were also milled alone. Binary physical mixtures (PM) composed of crystalline BOS and crystalline SIL were prepared as well. In the latter case, the ingredients were manually blended until a homogeneous mixture was formed (approx. 5 min).

### 2.2. Particle morphology

#### 2.2.1. Polarizing optical microscopy (POM)

The imaging was performed using a Nikon ECLIPSE LV100POL (Tokyo, Japan) polarizing microscope with a Fine Instruments WTMS-300 heating stage and a Canon camera coupled to a computer. Samples were placed between two coverslips (20 x 20 mm Menzel Gläser/ Thermo Scientific, Watham, MA, USA) and observations were carried out according to the following protocol:

1. Heating from 20 °C to 180 °C (for BOS samples only to 140 °C) at 5 °C min⁻¹.

2. Cooling to 20 °C at 10 °C min⁻¹.

3. Reheating as in step 1.

4. Cooling down as in step 2.

The images were captured only upon heating (points 1 and 3). The procedure was repeated twice for each sample. First, the polarizers were parallel. Second, the sample was observed between crossed polarizers (90°). The intensity of the light falling on the sample was the same during the entire procedure, and the images were captured always in exactly the same place. Microscopic images were processed using ImageJ v.1.53a software (NIH, USA).
### 2.2.2. Scanning electron microscopy (SEM) and particle size distribution (PSD)

The powder was adhered to a holder with double-sided copper tape. Its surface was coated with carbon using a 208 HR carbon sputter coater (Cressington Scientific Instruments, Watford, UK). Then, their morphology was analyzed using a Hitachi S-4700 (Japan) scanning electron microscope. The images were taken at a magnification of 100 x, 2 000 x, and 20 000 x. Then ImageJ v.1.53a software (NIH, USA) and OriginLab 2021b software were used to estimate PSD. In summary, at least 200 particles were selected from the images. Their circular equivalent diameters were calculated on the basis of particle area according to the monograph < 1776 > Image analysis of pharmaceutical systems published in the United States Pharmacopeia. The number-weighted PSD was converted into the volumetric PSD. They were presented as normal and cumulative curves. The median particle dimension ($d_{50}$) was determined on the basis of the cumulative distribution. This value ($d_{50}$) means that 50 % of the particles are smaller and 50 % of the particles are larger.

### 2.3. Powder X-ray diffraction (XRD)

A PanAnalytical X'Pert PRO MPD diffractometer (Almelo, Netherlands), equipped with X'Celerator detector, was used. The measurements were carried out at either high temperature or RT. When the analyzes were carried out at RT, the samples were placed into Linde-mann glass capillaries of 0.7 mm in diameter (Hilgenberg GmbH, Masfeld, Germany). Then, these capillaries were installed on a rotating sample holder to avoid any artifacts because of the preferential orientations of the crystallites. In turn, the high-temperature measurements were performed in Bragg-Brentano θ-θ geometry. The samples were placed in an Anton Paar TTK 450 chamber under vacuum. All samples were exposed to X-ray radiation (λ.Cu - Kα) with a wavelength of 1.540 Å. The diffractograms were recorded from 5 to 40° or 60°, with a counting time of 50 s/point and a measuring step of 0.0167°. X-ray diffraction patterns of crude BOS, crude SIL, and their recrystallized solid dispersion BM0.5 were analyzed at the 120 °C - 170 °C range by the Rietveld refinement in the FullProf program (Rodríguez-Carvajal, 1993). The fitting parameters included the scale factor, width, and shape parameters of the diffraction peaks, unit cell dimensions, preferred orientation, and background points. The crystal structures of crude BOS and crude SIL, the atomic coordinates, and temperature factors were fixed to values from the Cambridge Crystallographic Data Center (CCDC) database.

XRD patterns of samples stored in climate chambers were also collected in the Bragg-Brentano θ-θ geometry. The diffractograms were performed at RT from 2° to 40°. A counting time was 100 s/point and a 0.033° step was applied.

### 2.4. Differential scanning calorimetry (DSC)

The solid-state properties of binary formulations and single drugs were evaluated using a differential scanning calorimeter DSC Q1000 (TA

<caption>**Table 3**</caption>

The values of Sink Index for bosentan (BOS) and sildenafil base (SIL) in dissolution studies. SI > 3 is typical of sink conditions, whereas SI ≪ 3 indicates non-sink conditions.

<table><thead><tr><th rowspan="2">Dissolution solvent</th><th rowspan="2">Formulation name</th><th rowspan="2">Sample weighed for dissolution test [mg]</th><th colspan="2">Sink Index (SI)</th></tr><tr><th>BOS</th><th>SIL</th></tr></thead><tbody><tr><td rowspan="3">SGF</td><td>BM0.8</td><td>25</td><td>0.01</td><td>536</td></tr><tr><td>BM0.5</td><td>40</td><td>0.01</td><td>134</td></tr><tr><td>BM0.2</td><td>100</td><td>0.01</td><td>34</td></tr><tr><td rowspan="3">PBS</td><td>BM0.8</td><td>25</td><td>0.21</td><td>0.57</td></tr><tr><td>BM0.5</td><td>40</td><td>0.21</td><td>0.14</td></tr><tr><td>BM0.2</td><td>100</td><td>0.21</td><td>0.04</td></tr></tbody></table>

Instruments, Guyancourt, France) equipped with a refrigerated cooling system. Samples (3 – 5 mg) were analyzed in open cells (without lid). The ball milled samples were heated at 55 °C for 10 min before analysis to evaporate water. The first heating scans were performed from 10 °C to 190 °C. After cooling to 10 °C, the second heating scans were performed from 10 °C to 190 °C. All measurements were carried out in highly pure nitrogen (50 mL min⁻¹). The heating rate was 5 °C/min. Temperature and enthalpy readings were calibrated using pure indium at the same scan rate as that used in the experiments.

### 2.5. Fourier transform middle-infrared absorption measurements (FT-MIR)

FT-MIR measurements were performed in transmission mode using a Bruker VERTEX 70v vacuum spectrometer. The spectra were carried out in the spectral range of 370–4000 cm⁻¹ with a resolution of 2 cm⁻¹ and 32 scans per each spectrum. Powdered samples of crude components (BOS and SIL) as well as co-milled binary samples were mixed with KBr, compressed into pellets, and measured at room temperature. For the sake of comparison, binary physical mixtures were prepared and analyzed. Spectra were recorded using OPUS 7.0 software and presented with Origin 2017 Pro.

### 2.6. Frequency domain dielectric spectroscopy (FDDS)

Dielectric studies were performed using a Novocontrol Concept 81 instrument (Novocontrol Technologies GmbH & Co. KG Montabaur, Germany) impedance spectrometer set in the frequency range of 100 mHz – 1 MHz and measuring voltage of 0.3 V. Stabilization of temperature was ± 0.5 °C. The measurements of the solid dispersion BM0.5 were carried out during heating from 20 °C to 140 °C every 2 °C. The temperature range was chosen to register dielectric spectra in the glassy phase, close to the glass transition, and in the range that includes full recrystallization with $T_c$ (cold crystallization), but avoiding the start of melting because the DSC measurements clearly show that a relatively wide thermal anomaly associated with melting is very close to the end of a cold crystallization.

The samples were compressed into a 0.36 mm thick pellet and placed between two brass electrodes (9.6 mm in diameter) to form a flat capacitor. To ensure the stability of the system, it was placed on a Teflon ring.

### 2.7. Contact angle measurements

A drop shape analyzer DSA 25S (Krüss, Hamburg, Germany) and a Krüss Advance software v.1.7–01 were used for the measurements. The sessile drop technique was applied to determine the contact angle (wettability) formed between a drop of water (2 µL of MilliQ water) and the compact surface. Before analyses, the samples of co-milled powder (120 mg) were compacted using an Atlas Manual 15 T hydraulic press (Specac, Kent, UK) equipped with punches of 8 mm in diameter. The compaction force of 2 t was applied for 30 s. Then, a drop of water was metered on a compact deduced surface with a stainless steel needle of Ø = 0.5 mm. The drop images were captured for 3 s at 25 fps. An ellipse model was fitted to the drop contour to determine the contact angle. All measurements were made at 20 °C and RH = 28 %. Each sample was analyzed at least three times to ensure reproducibility.

### 2.8. Equilibrium solubility studies

The saturation solubility of BOS and SIL was determined by using an excess amount of each drug in a shake flask method. Distilled water, simulated gastric fluid (SGF) and phosphate buffer (PBS) of pH = 6.80 were used as solvents. The samples (n = 6) were thermostated at 37 °C ± 0.5 °C and shaken at 800 rpm using a uniThermix 2 pro (LLG Labware, Meckenheim, Germany) for 24 h. After dilution and filtration, the
concentration of dissolved drugs was determined using an HPLC-DAD method described below. Mean values (n = 6) ± standard deviations were calculated.

## 2.9. Drug dissolution studies

The *in vitro* concentration-time profiles of bosentan and sildenafil base dissolved from solid dispersions were studied using two different media, i.e. simulated gastric fluid (SGF, Ph. Eur. 10th Ed., pH = 1.20) without pepsin or phosphate buffer of pH = 6.80 (PBS). The values of Sink Index [SI (Schver and Lee, 2021; Sun et al., 2012)] for BOS and SIL respectively are given in Table 3. The tests were carried out at 37 °C ± 0.5 °C. An automated pharmacopoeial paddle dissolution apparatus was used, Hanson Research Dissolution Station Vision Elite 8 with an auto-sampler Visione AutoPlus™ Maximizer and a sample collector Auto-Fill™ device (Chatsworth, CA, USA) equipped in a set of small vessels (150 mL). Each test was carried out with samples corresponding to 20 mg of BOS and 100 mL of SGF or PBS. The paddle rotation speed was set at 75 rpm. Samples of 2 mL were withdrawn for 120 min and the same amount of replacement medium was added. The samples were transferred to test tubes containing 2 mL of ACN to prevent drug precipitation. Then, they were vortexed and filtered directly into HPLC vials. The concentrations of BOS and SIL dissolved were determined using the HPLC-DAD method described below. The mean values (n = 3) in µg mL⁻¹ and the corresponding standard deviations were calculated.

To better evaluate *in vitro*, the performance of amorphous formulations after oral administration, a two-stage dissolution was carried out using biorelevant media, namely fasted simulated gastric fluid (FaSSGF) and fasted simulated intestinal fluid (FaSSIF) of composition according to *Biorelevant.com*. The study was carried out at 37 °C ± 0.5° C, operating with the parameters described above. The difference was that in the acid stage, 75 mL of FaSSGF of pH = 1.60 was used. Under such conditions, the samples corresponding to 20 mg BOS were tested for 120 min. Then 25 mL of an aqueous solution composed of 0.48 g of sodium chloride, 0.34 g sodium phosphate anhydrous, 0.22 g of 3F powder (*Biorelevant.com*) and 0.09 g sodium hydroxide were added to each of the vessels in order to obtain 100 mL of FaSSIF of pH = 6.50 as a buffer stage solvent. This stage lasted 180 min, so the total test time was 300 min (5 h). The concentrations of BOS and SIL in the samples were determined using the HPLC-DAD method (see sec. 2.10). The mean values (n = 3) in µg mL⁻¹ and the corresponding standard deviations were calculated.

## 2.10. HPLC-DAD method

The concentrations of BOS and SIL dissolved in SGF or PBS were determined using an Agilent 1260 Infinity HPLC system (Waldbronn, Germany) connected to a diode array detector (DAD). Samples were filtered through nylon syringe filters (Ø = 0.22 µm). They were analyzed using an InfinityLab Poroshell 120EC-CN reversed phase LC column (4.6 x 100 mm; particle size 2.7 µm) with the corresponding guard column. The injection volume was 5 µL. The mobile phase was composed of ACN and 10 mM KH₂PO₄ mixed in a 40:60 (v/v) ratio (isocratic elution). The flow rate was 1.0 mL min⁻¹. The column oven temperature was set at 30 °C. The signals were detected at a wavelength of 267 nm. A mixture of ACN and DMSO (9 + 1) was used as a needle wash solvent. Run time was 3.50 min. Retention times were 2.20 min and 3.20 min for SIL and BOS respectively. When biorelevant fluids were used, the samples were filtered through polypropylene syringe filters (Ø = 0.22 µm). The composition of the mobile phase was modified and it was composed of 0.7 % TEA/PA (v/v) of pH = 3.0, ACN and MeOH mixed in a 58:17:25 (v/v) ratio (isocratic elution). Run time was 5.50 min. Retention times were 2 min and 4.70 min for SIL and BOS respectively. The calibration curve was prepared using crude BOS or crude SIL as standards to prepare stock solutions of 1 mg mL⁻¹ in ACN. A mixture of the dissolution solvent and ACN in a 1:1 (v/v) ratio was used to prepare dilutions. The

<figure><img src="image_2.png" /><figcaption>Fig. 1. XRD patterns of bosentan monohydrate (BOS)/sildenafil base (SIL) co-milled for 4 h. Bosentan fraction ranged from 0 to 1 as indicated in the sample name.</figcaption></figure>

linearity range together with LOD and LOQ is given in Table S1.

## 2.11. Statistical analysis

The ANOVA test was applied to evaluate differences in the values of the measured parameters. If the analysis of the variance showed a significant difference between groups, the Tukey post hoc test was performed to identify disparate groups. Significance was assumed if p-value was below 0.05. Statistical analyzes were carried out using Origin software v. 2021b (OriginLab Corp., Northampton, MA, USA).

# 3. Results and discussion

## 3.1. Crystallinity and thermal properties of bosentan-sildenafil solid dispersions

Fig. 1 shows diffractograms recorded under ambient conditions for both crude drugs, i.e. BOS and SIL and their co-milled solid dispersions (BM).

The diffractograms of crude BOS and SIL (form I) confirm their crystalline P2₁/c structures reported in the literature (Barbas et al., 2018; Kaur et al., 2013). Regardless of the weight ratio of the compounds, the diffractograms of all co-milled formulations (BM0.2, BM0.5, BM0.8) clearly show the disappearance of sharp diffraction peaks, which after their co-processing are replaced by a 'halo' pattern typical of the amorphous phase. However, on the basis of these diffractograms, it cannot be concluded if there are physical mixtures of the amorphous forms of each of these drugs or if they form homogeneous glassy solutions.

The heat flow curves of the physical mixtures loaded with BOS and SIL are presented in Fig. 2a, whereas those typical of their quenched liquids or co-milled solid dispersions are presented in Fig. 2b and Fig. 3 respectively. For the sake of comparison, the thermal properties of crude SIL are shown in Figs. S1-S4 and those of crude BOS have already been described in detail in our previous research (Krupa et al., 2022).

In the DSC thermograms of the physical mixtures (Fig. 2a), a wide endotherm corresponding to BOS dehydration is followed by its melting endotherm. The third endotherm shows the dissolution of SIL in liquid BOS. The end of this endotherm marks the end of the dissolution and is the most shifted to lower temperatures for PM0.8 loaded with the lowest amount of SIL (compared to the crude drug). In turn, the heat flow curves of quenched liquids (Fig. 2b) provide evidence of co-
<figure><img src="image_3.png" /><figcaption>Fig. 2. Heating (5 °C/min) DSC scans of binary formulations composed of bosentan monohydrate (BOS) / sildenafil base (SIL) in comparison to crude drugs: (a) physical mixtures (PM), (b) quenched-liquids (QL), (c) co-milled samples. Bosentan fraction ranged from 0 to 1 as indicated in abbreviated sample name. DSC scans of bosentan anhydrous and BM samples were recorded after 15 min of annealing at 55 °C to remove water.</figcaption></figure>

amorphization of both crystalline drugs. Importantly, under this thermal co-processing homogeneous molecular alloys are formed as there is only one glass transition. The midpoint shifts reflecting the composition of the binary system. It moves toward higher temperatures with an increasing bosentan load. Importantly, there has been no recrystallization of the amorphous form while heating up to 190 °C (5 °C min⁻¹) if the SIL load ranges between 20 (PM0.8) and 50 (PM0.5) wt. %, but when it increases from 50 to 80 wt% (PM0.2), a wide exotherm indicating crystallization appears. This shows that the SIL load in this binary system is higher than its solubility in BOS. In consequence, SIL crystallizes to reach saturation, then it gradually dissolves under further heating, which is illustrated by a dissolution endotherm. The location of this endotherm is similar to that recorded for SIL in the physical mixture PM0.2 (Fig. 2a). The thermal properties of high energy ball milled solid dispersions are presented in Fig. 2c. They are similar to those previously described for quenched liquids. However, the kind of amorphization method, i.e. thermal treatment or mechanical activation, has an impact on the physical stability of the amorphous product. The ball milled amorphous solid dispersions are slightly more susceptible to recrystallization than the corresponding quenched liquids. Among them, the most stable is that loaded with the greatest amount of BOS, i.e., 80 wt% (BM0.8). Its heating up to 190 °C does not cause crystallization which is observed if the BOS load is lower, e.g. 50 wt% in BM0.5 or 20 wt% in BM0.2. Interestingly, the quenched liquid obtained from BM0.5 does not crystallize upon heating (data not shown).

To identify the composition of the crystalline phase that appears under heating of co-amorphous solid dispersions BM0.5 and BM0.2, XRD patterns were recorded as a function of temperature and they were compared with those of single components (Fig. 3). This comparison indicates that SIL at least partially crystallized upon heating from its solution in liquid (melted) bosentan, as the Bragg peaks typical of its crystalline structure are visible in the diffractograms shown in Fig. 3. In turn, there is no trace of crystalline BOS peaks. This indicates that SIL undergoes cold crystallization, while BOS retains its amorphous form. Since SIL can exist in two polymorphic forms: I and II [anhydrous (Barbas et al., 2018), Rietveld analysis was applied for the refinement of the structure of the crystallized drug (Rodríguez-Carvajal, 1993). The fitting parameters included the scale factor, width, and shape parameters of the diffraction peaks, unit cell dimensions, preferred orientation, and background points. The results of the Rietveld analysis confirmed that heating of BM0.5 resulted only in crystallization of SIL to its original form I (Fig. S5, S6).

Additionally, the crystallinity of the amorphous solid dispersions, showing the most favorable thermal properties, i.e. BM0.5 and BM0.8, was also evaluated after storage for two weeks under standard (25 °C/ 60 % of RH) or accelerated conditions (40 °C/75 % RH). X-ray diffraction patterns recorded at RT are presented in Fig. 4. They clearly show that BM0.5 and BM0.8 are XRD-amorphous after two weeks of storage at 25 °C/60 % RH. However, some traces of crystallinity appear in BM0.8 under accelerated conditions, but they are invisible in BM0.5. These results suggest that the co-processing of BOS and SIL with a polymer, preferably of high glass transition temperature, e.g. copovidone, might be necessary to fully stabilize the amorphous form.

To evaluate the strength of interactions between BOS and SIL in their binary glassy solutions and then to be able to calculate the Tg for any component ratio, the experimental data (Tg) recorded for representative binary formulations and those of single compounds are plotted in Fig. 5. The data is fitted by the Gordon-Taylor (GT) equation (Eq. (1)):

$$Tg(X_{BOS}) = \frac{[X_{BOS} \cdot Tg^{BOS} + K \cdot (1 - X_{BOS}) \cdot Tg^{SIL}]}{[X_{BOS} + K \cdot (1 - X_{BOS})]} \quad (1)$$

where X_BOS is the weight fraction of BOS in the formulation, TgBOS and TgSIL are, respectively, the glass transition temperatures of crude BOS and crude SIL, and K is a fitting parameter, describing the curvature of the evolution.
<figure><img src="image_4.png" /><figcaption>Fig. 3. Diffractograms of co-amorphous solid dispersions prepared by high energy ball milling: (a) BM0.5 and (b) BM0.2 registered in various temperatures (5 °C/min). For comparison, XRD-patterns of either binary formulations or single components determined at room temperature (RT) are also shown.</figcaption></figure>

The best fit is obtained for $K = 0.85 \pm 0.01$, which is close to the $K_{id}$ value, that is, 0.89, expected for an ideal mixture (Eq. (2)):

$$K_{id} = \Delta Cp^{SIL} / \Delta Cp^{BOS} = 0.42 (\text{J/g} \cdot {}^{\circ}\text{C}) / 0.47 (\text{J/g} \cdot {}^{\circ}\text{C}) = 0.89 \quad (2)$$

where $\Delta Cp^{SIL}$ and $\Delta Cp^{BOS}$ is a difference between the specific heat capacity (Cp) of the supercooled liquid and that of the crystal measured just above Tg of SIL and BOS, respectively.

## 3.2. Impact of heating on the morphology of SIL, BOS and their co-milled solid dispersions

Fig. 6 shows microscopic images recorded in polarized light during the first heating (5 °C min⁻¹, see sec. 2.2.1) of crude BOS, crude SIL, and their solid dispersion BM0.5 prepared by co-milling. It can be seen that the heating of BOS results in melting of the drug particles which starts

around 100 °C (Fig. 6 a-h). The SIL particles remain unchanged at this temperature (Fig. 6 i-p) as they melt only at around 180° C (data not shown), which is perfectly in line with the DSC results shown in Fig. 2. For the sake of comparison, the images recorded upon heating of SIL quenched liquid are shown in Figure S7. When heated for the first time, this sample produces round, regular shapes with different radii. The droplets of SIL do not change shape or position when heated to the melting point, but their surface changes. The drops have a very smooth surface at 70 °C, then they become wrinkled (Fig. S7 b-d). Apart from wrinkles on the surface of the drops, their darkening is also observed with further heating (Fig. S7 c,d). The formation of the wrinkles on the surface of SIL sample has already been reported (Kolodziejczyk et al., 2014). The authors explained this phenomenon by the formation of hydrogen bonds between SIL molecules which preceded devitrification. We believe that the wrinkles and creases we observe have the same
<figure><img src="image_5.png" /><figcaption>Fig. 4. XRD patterns of co-milled binary formulations BM0.5 and BM0.8 recorded at RT after their storage in climate chambers (Memmert HPP108, Schwabach, Germany) at 25 °C/60 % RH or at 40 °C/75 % RH for 2 weeks. The powder was placed in tightly closed glass vials with desiccators.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 5. Gordon-Taylor (GT) plot showing variations of Tg in bosentan-sildenafil base co-amorphous solid dispersions prepared using high energy ball milling. Fitting of the experimental curve by GT equation is shown as a solid red line (r² = 0.999).</figcaption></figure>

origin. Upon re-heating of its quenched liquid, the droplets are not visible, but the crystallization starts from about 90 °C together with the isotropic growth of the crystal phase in all directions (Fig. S7 j-l). In contrast to the first heating, there are no visible wrinkles.

The images recorded for solid dispersion BM0.5 while heating are shown in Fig. 6 q-z and Fig. 6 q1-z1 for a single and crossed polarizers, respectively. Similarly to crude BOS and SIL, these images are also consistent with the DSC results (Fig. 2). The sample is amorphous up to 130 °C, then a crystalline phase appears (Fig. 6 y1), but it gradually disappears under further heating (Fig. 6 z1). When the quenched liquid is analyzed, the amorphous state persists throughout the temperature range and crystallization is not visible (data not shown).

### 3.3. Impact of co-milling on the physical stability of the amorphous solid dispersion BM0.5

The solid dispersion BM0.5 in which BOS and SIL are combined

together in equal wt. parts (1:1) was chosen to assess the influence of each of the single components on the physical stability of their co-milled amorphous solid dispersion. This sample is amorphous at the beginning of the dielectric measurements, but the following phase transitions occur while heating: glass → intermediate state → crystal → liquid. The number of relaxation processes depends on the temperature range. There is only one process with relaxation frequency ca. 0.1 MHz at a temperature ranging from 20 °C to 48 °C (in the middle of the glassy phase), while no relaxation is detected between 50 °C and 74 °C, and a relaxation process is detected again in the range of 76–94 °C. In turn, two relaxation processes are recorded at 96–140 °C. The Havriliak-Negami (HN) model with the DC specific electric conductivity term was fitted to the experimental data ε'(f) and ε''(f) described by Eq. (3):

$$ \epsilon''(f) = \epsilon'(f) - i\epsilon''(f) = \epsilon_{\infty} + \sum_{i=1}^{k} \frac{\Delta\epsilon_i}{(1 + (2\pi if\tau_{HN})^{a_i})^{b_i}} - i \frac{\sigma_{DC}}{2\pi f\epsilon_0} \quad (3) $$

where: $\epsilon_{\infty}$ – permittivity at high frequency limit, $\Delta\epsilon$ – dielectric increment, $f$ – frequency, $\tau_{HN} = 1/(2\pi f_0)$ – relaxation time, $a$ and $b$ – parameters of symmetrical and asymmetrical broadening of the spectra, $\epsilon_0$ – vacuum permittivity, $\sigma_{DC}$ – DC specific electric conductivity.

Fig. 7 presents the dielectric absorption measured for BM0.5 in several selected temperatures together with the result of fitting the HN model (the fitting parameters are shown as an example in Fig. 7 c). A high-frequency relaxation is the structural $\alpha$-relaxation while that of a low-frequency is called $\alpha'$ relaxation. It is clearly visible that both relaxation processes shift toward higher frequencies with an increasing temperature (Fig. 7). The relaxation time $\tau_{HN}$ was converted to the proper relaxation time $\tau_r$, according to Eq. (4):

$$ \tau_r = \frac{1}{2\pi f_r} = \tau_{HN} \left[ \sin\left(\frac{\pi a}{2+2b}\right) \right]^{1/a} \left[ \sin\left(\frac{\pi ab}{2+2b}\right) \right]^{-1/a} \quad (4) $$

In the glassy and intermediate phases structural $\alpha$-relaxation can be observed (Kolodziejczyk et al., 2013; Minecka et al., 2022). An intermediate phase in amorphous SIL is described as a supercooled liquid (Kolodziejczyk et al., 2013). In our case, we did not observe any liquid features in the temperature range of the intermediate phase typical of this binary amorphous solid dispersion (BM0.5). In the glassy phase, structural $\alpha$-relaxation is beyond the measurement range of conventional spectrometers (the relaxation time is too long) and only secondary relaxation can be registered, e.g. activation $\beta$-relaxation, which is associated with small angular reorientations of the whole molecule or the fragments of the molecule. In amorphous SIL, a secondary relaxation process below Tg whose relaxation frequency increased with increasing temperature has been described (Kolodziejczyk et al., 2013). Measurements were carried out in a wide temperature range from approximately –91 °C to 35 °C (1000/T = (5.50–3.25)1/K) and the relaxation process was interpreted as $\beta$-relaxation with an activation energy of 50 kJ/mol. In our case, the measurements were performed upon heating the sample from 20 °C and the relaxation frequency of the process recorded for BM0.5 in the glassy phase practically does not depend on the temperature, so it is a non-activated process. In this temperature range, the $\beta$-relaxation time typical of SIL also changed slightly with temperature (Kolodziejczyk et al., 2013). Taking into account the findings as well as the low dielectric absorption $\epsilon''$ for both SIL and BM0.5, we interpret the process registered for solid dispersion (BM0.5) as $\beta$-relaxation. It should be noted, however; that the relaxation time of this process is practically constant upon heating, which may prove some kind of stabilization of this molecular relaxation in the presence of BOS. To fully confirm our hypothesis, we plan to study amorphous solid dispersions with various drug ratios. Furthermore, by comparing the relaxation time ($\beta$-relaxation) determined at 35 °C for SIL (3.16●10⁻⁷ s) and BM0.5 (1.81●10⁻⁶ s), it can be concluded that bosentan slows relaxation by about 5.73 times. In general, the presence of BOS not only stabilizes the molecular mobility of this process but also slows it.
<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Images captured in a polarizing light microscopy under heating for: crude BOS (a-h); crude SIL (i-p); their co-milled solid dispersion BM0.5 (q-z1). A single polarizer (in odd rows) or crossed polarizers (in even rows). Inset scale bar corresponds to 100 µm.</figcaption></figure>

Above the Tg of BM0.5, structural α-relaxation is recorded, which is much stronger than β-relaxation. Fig. 8 shows the registered α-relaxation process. The relaxation frequency $f_{HN}$ increases with increasing temperature, which induces an increase in the molecular mobility of BM0.5. The contribution of specific electric conductivity related to translational motions of ions is also visible, especially at higher temperatures and low frequencies, which has also been reported for SIL (Kolodziejczyk et al., 2013).

In general, α-relaxation is related to the glass transition and is called the dynamic glass transition. In turn, the static glass transition and the associated Tg are determined from DSC measurements and correspond to a frequency of about 1 mHz. From the molecular point of view,

α-relaxation in the case of polymers is related to the micro-Brownian motion of chain segments (conformational changes). However, the mechanisms responsible for this dissipative process in the case of materials with low molecular weight that exhibit the glass transition, such as BOS or SIL, are not fully understood. Nevertheless, because of their cooperative nature, they can be related to the micro-Brownian motions of whole molecules.

As to β-relaxation, it has only been registered at a few of the highest glassy phase temperatures. On the other hand, α-relaxation is visible in a wide temperature range (76–140 °C) and the relaxation time of this process as a function of the reciprocal temperature shows typical non-linear behavior (Fig. 9 a). Because this is a non-Arrhenius behavior (in
<figure><img src="image_8.png" /><figcaption>Fig. 7. Dielectric absorption (points) with the results of fitting Havriliak-Negami model (solid lines) for BM0.5 measured at 100 °C (a), 120 °C (b), 130 °C (c) and 140 °C (d). The legend shown in (d) corresponds to all graphs.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Fig. 8. Dielectric dispersion ε''(f) registered for BM0.5 at 76 – 94 °C (a), 96 – 112 °C (c) and 114 – 140 °C (d). For better visibility, enlarged area of shifted α-relaxation maximum with increasing temperature are shown in (b) and insets in (c) and (d).</figcaption></figure>

the intermediate phase), the Vogel-Fulcher-Tammann (VFT) equation (Eq. (5)) was used to describe the temperature dependence of the relaxation time τr,α:

$$ \tau_{r,\alpha}(T) = \tau_{\infty} \exp \left( \frac{A}{T - T_{VFT}} \right) \qquad (5) $$
<figure><img src="image_10.png" /><figcaption>**Fig. 9.** Dependence of relaxation time on reciprocal temperature (symbols) for all processes (a) and Angell plot (b). Solid lines in (a) result from fitting Vogel-Fulcher-Tammann equation for α-relaxation. Dashed ellipse in (a) marks area where crystallization starts; small continuous ellipse surrounds point where extrapolated fit intersects $log10τr = 2$ ($τ_{max} = 100$ s) and determines Tg. Inset in (b) presents determination of kinetic fragility.</figcaption></figure>

$$log_{10}τ_{r,a}(T) = log_{10}τ_{∞} + \frac{A}{T - T_{VFT}}log_{10}e \quad (6)$$

where $τ_{∞}$, $T_{VFT}$ and A are fitting parameters. The $log10τ_{∞}$ Eq. (6), $T_{VFT}$ parameters obtained from fitting Eq. (6) to the experimental data are equal to -10.52(0.53), 309(3) K and -17.05(0.50), 254(3) K (Kolodziejczyk et al., 2013) for BM0.5 and SIL, respectively. Thus, it can be concluded that the addition of BOS increases the TVFT temperature.

The dielectric spectra registered as a function of temperature for BM0.5 indicate a phase transition around 104 °C, which is in agreement with the DSC results (ca. 100 °C crystallization begins). In the temperature range of 104–112 °C, the temperature relationship of the relaxation time differs from the typical VFT dependence (Fig. 9 a), while for temperatures equal to or higher than 112 °C, it is non-Arrhenius and, therefore, can be described by the Vogel-Fulcher-Tammann equation. In our opinion, the high-frequency process registered in both the transition and the crystal phases is α-relaxation, but it occurs much faster than that at lower temperatures. The $log10τ_{∞}$, $T_{VFT}$ parameters obtained from the fitting procedure in this temperature region are equal to -15.36(0.50) and 279.5(5.6) K, respectively, and are close to those obtained for SIL (Kolodziejczyk et al., 2013).

Another relaxation process with a frequency lower than α-relaxation is registered in the temperature range of 104–140 °C. The typical relaxation time of SIL sample is in the range of 1.78 s – 0.10 ms for 1000/T = 2.90–2.75 (Kolodziejczyk et al., 2013), while it ranges from 3.16 s to 0.01 ms for 1000/T = 2.80–2.55 for BOS (Minecka et al., 2022). In the present study, the relaxation time is in the range of 23.89 s – 0.41 ms (1000/T = 2.86–2.65) for the binary amorphous solid dispersion (BM0.5). Thus, it can be concluded that BOS delays this relaxation relative to the SIL sample.

This additional relaxation process in the low frequency range is registered when crystallization begins. It is known that with the beginning of crystallization and during a further increase in temperature, the degree of crystallinity increases, whereas the level of the amorphous form decreases. Here, the degree of crystallinity is defined as a ratio of the mass of the crystallized part to the total mass of the sample. For BM0.5, ε' and $Δε$ for α-relaxation drastically decrease with increasing temperature from approximately 95 °C (Fig. S8). The same has been observed for SIL alone and in its binary physical mixtures with polymers (Knapik-Kowalczuk et al., 2020; Kolodziejczyk et al., 2013). The dielectric increment $Δε$ is proportional to the number of molecules N, each with a dipole moment $μ$ ($Δε \sim Nμ^2$). Taking into account a decreasing number of reorienting dipoles as crystallization progresses,

reduced $Δε$ reflects the beginning of crystallization. In turn, the low-frequency process, denoted as α', may be related to the conformational mobility derived from the presence of a certain amount of the amorphous fraction that adheres to the surface of previously formed crystals. This relaxation process takes place until crystallization has been finished, and its relaxation time decreases with an increase in temperature by three orders of magnitude. As can be seen in Fig. 9 a, two temperature regions for the α'-relaxation can also be distinguished, namely 104–120 °C and 122–140 °C, which, based on the DSC results, correspond well to almost the entire anomaly associated with the crystallization process and its final descending part, respectively. There could be a certain threshold degree of crystallinity (close to 1), beyond which a certain fluctuation is observed at the molecular level. Although the character of the α'-relaxation seems to be non-Arrhenius. The Vogel-Fulcher-Tammann equation is also not suitable for describing it. This phenomenon has been reported for SIL (Kolodziejczyk et al., 2013), but not for BOS. Further studies are necessary to fully understand the mechanism of this relaxation, e.g. infrared studies as a function of temperature.

An important parameter that characterizes structural α-relaxation is fragility. This parameter indicates how quickly the dynamics of the material is slowed as it cools down to the Tg. The most common definition of fragility is the kinetic fragility index $m_p$, which describes the temperature dependence of the relaxation time near the glass transition and is expressed by Eq. (7):

$$m_p = \left( \frac{dlog_{10}τ_{r,a}}{d(T_g/T)} \right)_{T=T_g} \quad (7)$$

Glass-formers can exhibit various $m_p$ values. When $m_p$ is large, they are called ‘fragile’ materials, but if $m_p$ is small, they are classified as ‘strong’ materials (Angell, 1995). In fragile materials, the molecular mobility changes rapidly at temperatures close to Tg, which may be associated with their poor thermodynamic stability and consequently, greater tendency to crystallize. The value of $m_p$ for BM0.5 is 120.31 at $τ_{r,a} = 100$ s (Fig. 9 b), indicating that this is a fragile material. In turn, SIL is classified as a medium fragile material with $m_p = 85$ (Kolodziejczyk et al., 2013). Thus, the co-milling of BOS with SIL (BM0.5) leads to an increase in the fragility of this binary co-amorphous system, but at the same time, thanks to the presence of BOS, the value of Tg increases, and finally, the formulation should be stable under ambient conditions.
<figure><img src="image_11.png" /><figcaption>**Fig. 10.** Particle size distribution in high energy ball milled solid dispersions loaded with bosentan monohydrate and sildenafil base: (a) normal particle size distribution curves, (b) cumulative particle size distribution curves, black dash line and labels show d₅₀.</figcaption></figure>

### 3.4. Impact of co-milling on chemical interactions between the compounds

FT-MIR spectra of co-milled formulations and those of physical mixtures recorded under ambient conditions are presented in Figure S9. They do not show any specific interactions between BOS and SIL induced by mechanical activation. There is only the broadening of the bands characteristic to functional groups of single compounds (Krupa et al., 2017), which reflects the disorder in their structure caused by mechanical activation. However, there is no creation of new bands.

### 3.5. Impact of milling on particle size, surface wettability, and drug dissolution

The impact of high energy ball milling on the particle size of crude drugs and their binary solid dispersions is presented in Fig. 10. Their particle size distribution is bimodal or multimodal, but at the same time, the mean particle size is considerably reduced after that processing. For example, the typical value of d₅₀ of crude BOS is 209 µm, which is almost ten times higher than that of crude SIL, i.e. 23 µm. After high energy ball milling of single drugs, their particle size is reduced to 7 µm or 8 µm respectively. The value of d₅₀ for co-amorphous solid dispersions ranges from 7 µm to 14 µm and is the highest for BM0.8 in which BOS load prevails (Fig. 10).

<figure><img src="image_12.png" /><figcaption>**Fig. 11.** Mean contact angle (±standard deviations) determined immediately after water drop metering on compact surface (n = 3).</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Fig. 12.** Ionisation of: (a) bosentan monohydrate and (b) sildenafil base in solvents used for drug release studies. Simulated gastric fluid (SGF, pH = 1.20) and phosphate buffer (PBS, pH = 6.80), Sink Index (SI).</figcaption></figure>

The amorphization of BOS and SIL upon high energy ball milling makes the surface of particles significantly (p < 0.05) more hydrophilic compared to crude crystalline drugs (Fig. 11). The mean contact angle is reduced of about 10°. There is no significant difference (p > 0.05) in wettability between amorphous SIL (BM0) and amorphous BOS (BM1.0), as their contact angle is approximately 62°. However, the mean contact angle of co-milled binary formulations ranges from 65° to 69° and is lower than that of crystalline compounds, but slightly higher than that of single amorphous drugs. Research on celecoxib (Puri et al., 2010) provided the evidence that the number of polar groups on the surface of the amorphous material could be greater than that observed for crystals, and therefore; the degree of interactions with water molecules via hydrogen bonding might be higher. Moreover, high compaction pressure applied on the bed of the amorphous co-milled powder that shows conformational flexibility within short range order may contribute to further spatial rearrangement of chemical groups and creation of new polar or nonpolar chemical interactions between neighboring molecules.

Figure S10 shows the results of equilibrium solubility studies carried out in distilled water and buffers that simulate pH variations in the GIT.
<figure><img src="image_14.png" /><figcaption>**Fig. 13.** *In vitro* concentration–time profiles recorded for bosentan (BOS, in black) and sildenafil base (SIL, in blue) in PBS of pH = 6.80: (a) BM0.2, (b) BM0.5, (c) BM0.8. Solid squares show performance of binary solid dispersions; open circles correspond to physical mixtures. Dash lines show equilibrium solubility of bosentan monohydrate (in grey) and sildenafil base (in blue) in PBS at 37 °C.</figcaption></figure>

In addition, a scheme illustrating the percentage of ionisation of BOS and SIL in a function of pH is presented in Fig. 12. Since BOS pKa is 5.46, this drug is mostly un-ionised at pH < 3.46. As a consequence, it is impossible to accurately determine its saturation solubility in SGF where its signal is below the LOQ (1.97 µg mL⁻¹, Tab. S1) of the HPLC-DAD method. On the other hand, the solubility with BOS determined in PBS of pH = 6.80 is the highest (41.56 ± 0.48 µg mL⁻¹), because here the drug is almost fully ionised (>95 %). In contrast to BOS, the equilibrium solubility of SIL (weak) base of pKa = 5.99 in pH = 6.80 is almost 1000 times smaller (28.61 ± 12.88 µg mL⁻¹) than that determined in SGF (26.78 ± 3.49 mg mL⁻¹). These results confirm that the solubility of BOS and SIL is pH-dependent, but even if the equilibrium solubility of these

drugs is evaluated in distilled water, it differs significantly between these compounds (p < 0.05). The solubility of SIL (53.90 ± 9.92 µg mL⁻¹) is more than twenty times greater than that of BOS (2.64 ± 0.10 µg mL⁻¹).

In Figure S11, the *in vitro* concentration–time profiles of BOS and SIL recorded in simulated gastric fluid (SGF, pH = 1.20) are presented, while those recorded in phosphate buffer (PBS, pH = 6.80) that mimics intestinal conditions are shown in Fig. 13.

Since the solubility of the drugs is pH-dependent, the Sink Index (SI) values vary significantly in SGF and PBS (Tab. 3, Fig. 12). Due to the fact that SIL is fully ionised in SGF, SI is greater than 3, indicating that SIL dissolution is studied under sink conditions. Taking into account that the
<figure><img src="image_15.png" /><figcaption>**Fig. 14.** *In vitro* concentration–time profiles recorded for BOS (a) and SIL (b) in two stage dissolution test where after 120 min FaSSGF of pH = 1.60 was changed to FaSSIF of pH = 6.50. Dash lines show equilibrium solubility.</figcaption></figure>

equilibrium solubility of SIL in SGF is 26.78 $mg mL^{-1}$ (Fig. S10), the dissolution of SIL from solid dispersions BM0.5 and BM0.8 is immediate (Fig. S11). The same is true for physical mixtures. Moreover, monitoring of drug release from BM0.2 (80 wt% of SIL) is difficult as the concentration of SIL is so high, even after the first 5 min of the study, that samples require a significant dilution. Therefore, the signal from un-ionised BOS that has equilibrium solubility in SGF < 2 $µg mL^{-1}$ (SI ≪ 3, non-sink) is far below LOD (0.67 $µg mL^{-1}$). For that reason, BOS supersaturation in SGF could only be evaluated for BM0.5 and BM0.8 (20–50 wt% of SIL). It was stated that BOS co-amorphization with SIL enables to rapidly reach BOS supersaturation (Fig. S11). However, the maximum concentration is reached after 5–10 min, and then, it decreases dramatically. A significant increase (p < 0.001) in the area under the *in vitro* BOS concentration–time curve with maximal concentrations at supersaturation ranging between 3.13 ± 0.10 $µg mL^{-1}$ and 4.94 ± 0.17 $µg mL^{-1}$ is visible when the SIL load increases from 20 to 50 wt% (Fig. S12).

The dissolution of BOS and SIL in PBS at pH = 6.80 is shown in Fig. 13. Herein, the performance of both drugs is studied under non-sink conditions (SI ≪ 3). BOS is almost fully ionised (95.23 %), whereas the

ionisation of SIL is low, that is, 13.70 %. When the SIL load in the amorphous formulation ranges from 20-50 wt%, long lasting supersaturated BOS solutions form rapidly (Fig. 13 b,c). The highest level of BOS supersaturation is reached for BM0.8 (20 wt% of SIL) and it is twice that of BM0.5 (50 wt% of SIL). In contrast to these two formulations mentioned above, there is no supersaturation when BM0.2 is studied, and the shape of *in vitro* concentration–time profiles resembles that of physical mixtures (Fig. 13 a). The sample weight used for the dissolution studies always corresponds to 20 mg of BOS (200 $µg/mL$) so the load of SIL varies from 5 mg (BM0.8) to 80 mg (BM0.2). SIL saturation in PBS is reached only when BM0.8 is studied. In turn, the concentrations of SIL dissolved in BM0.2 and BM0.5 are both below saturation (Fig. 13).

To better assess *in vitro* performance of the amorphous formulations after oral administration, two stage dissolution was also carried out using biorelevant fluids, i.e. FaSSGF shift to FaSSIF (Fig. 14). In the acid stage (FaSSGF of pH = 1.60), the shape of the *in vitro* concentration–time profile of both drugs is similar to that recorded in SGF (Fig. S11). The BOS reaches supersaturation after 5–10 min. Then, its concentration decreases to the saturation level. At the same time, the dissolution profile of SIL is typical of formulations of immediate release. However,
the change in pH from 1.60 to 6.50 reveals that BOS slowly precipitates from its supersaturated solution formed in FaSSIF and therefore; its concentration gradually decreases for 3 h. This is not observed if PBS of pH = 6.80 is used as a dissolution medium, which suggests that surfactants, namely lecithin & bile salts, present in FaSSIF can delay precipitation, but they are unable to fully stop it (Fig. 14 a). Moreover, the precipitation of SIL previously dissolved in FaSSGF is also visible. This phenomenon is particularly pronounced if the SIL load in the amorphous solid dispersion is high (Fig. 14 b).

## 4. Conclusions

Throughout recent years various drug combinations have been investigated with the aim of enhancing the effectiveness of therapy and improving patient compliance (Wilkins et al., 2024). In this study, the properties of co-milled molecular alloys formed between BOS and SIL have been described for the first time. The glass transition temperatures of these co-amorphous formulations are relatively high and range from 64 to 78 °C, which suggests that these formulations should remain in the glassy state upon storage under ambient conditions. The results of XRD and DSC studies supported this conclusion and they revealed that the thermal stability of co-amorphous binary systems is strongly related to the SIL load which, in contrast to the BOS, shows a strong tendency to crystallize upon heating. This risk is particularly high when the SIL load is 50 wt% (BM0.5) or greater (BM0.2).

The solubility of BOS and SIL is pH-dependent, and they can be considered as a combination of a weak acid and a weak base, respectively. Thus, SIL is ionised in the gastric fluid and dissolves rapidly. However, it is un-ionised at intestinal pH (PBS) and dissolves slowly, forming unsaturated solutions after 120 min. Only when SIL is combined with an excess of BOS is the saturation level reached. In turn, the co-amorphization of BOS with SIL leads to long-lasting supersaturated BOS solutions in either SGF or PBS with maximal concentrations up to three times higher than those typical of equilibrium. This effect is the most pronounced for BM0.8 and BM0.5. The application of biorelevant fluids in a dynamic two-stage dissolution study confirmed that BM0.5 and BM0.8 are capable of creating supersaturation of BOS in the GIT which lasts for a few hours. Moreover, two-stage dissolution also provided the evidence that SIL can precipitate when pH increases to 6.50 and its ionisation is reduced.

In conclusion, the results of dissolution studies and those of preliminary stability tests carried out under normal (25 °C/60 % RH) and accelerated (40 °C/75 % RH) storage conditions indicate that a further optimization of these binary amorphous solid dispersions should include the mixture of drugs with a polymeric matrix-forming excipient, e.g. copovidone, that could better prevent their precipitation in an aqueous environment and ensure long-term stability.

Finally, the drug ratio in the best performing solid dispersions, that is, BM0.5 and BM0.8 is close to a new dose regimen proposed by clinicians (Amin et al., 2015; Li et al., 2021), in which BOS is administered orally twice a day (BID) at low doses, that is, 62.5 mg instead of 125 mg together with 50 mg of SIL (BID).

### CRediT authorship contribution statement

**Dominik Strojewski:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. **Sebastian Lalik:** Visualization, Investigation, Formal analysis. **Florence Danède:** Investigation. **Natalia Górska:** Visualization, Investigation. **Aleksandra Deptuch:** Visualization, Data curation. **Monika Marzec:** Writing – review & editing, Validation, Supervision, Methodology. **Jean-François Willart:** Writing – review & editing, Validation, Supervision, Methodology, Conceptualization. **Anna Krupa:** Writing – review & editing, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

### Declaration of competing interest

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.

### Data availability

Data will be made available on request.

### Acknowledgements

The research was carried out thanks to Sonata Bis grant no DEC-2019/34/E/NZ7/00245 financed by the National Science Centre in Poland and BGF-Séjour scientifique ed. 2020 grant funded by the French Embassy in Poland on behalf of the French Government. This research was supported in part by the Excellence Initiative, Research University Program at Jagiellonian University in Kraków (ID.UJ). Research was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08).

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2024.124312.

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