Selective Formation of Coamorphous Systems with Enzalutamide: Benzene Rings as Key Structural Features

Selective formation of coamorphous systems with enzalutamide: Benzene rings as key structural features Argyro Chatziadi * , Kateˇ rina Neubergerov´ a , Venkata Krishna Rao Balaga, Dan Trunov , Miroslav ˇ So´ oˇ s * Department of Chemical Engineering, University of Chemistry and Technology in Prague, Technick´ a 3, 16628, Prague 6, Czech Republic ARTICLE INFO Keywords: Coamorphous Enzalutamide Aromaticity Mechanochemistry Ball-milling Dissolution ABSTRACT Enzalutamide (ENZ), a non-steroidal antiandrogen used in the treatment of metastatic castration-resistant prostate cancer, exhibits poor aqueous solubility and bioavailability. Coamorphous systems, formed between an active pharmaceutical ingredient (API) and a low-molecular-weight coformer, offer a promising strategy to enhance solubility and dissolution. Here, we systematically screened a range of small organic acids and amino acids as coformers for coamorphous system formation with ENZ. Interestingly, full coamorphous systems formed exclusively with coformers containing a benzene ring, including benzoic acid, salicylic acid, 2-aminobenzoic acid, 2,5-dihydroxybenzoic acid (25H), L-phenylalanine, and L-tryptophan. Crystal structure analysis revealed that ENZ is stabilized in its crystalline state through strong π – π stacking, but these interactions are weakened or disrupted upon amorphization. Coamorphization is facilitated by coformers that can stabilize the amorphous phase through molecular complementarity and favorable packing. FTIR indicated limited new strong interactions, suggesting stabilization mainly via molecular mixing and weak π – π contacts. Thermal analysis confirmed single-phase systems with distinct T g values, and stability studies revealed that the ENZ–25H system remained amorphous for nearly four months, outperforming others. Dissolution testing demonstrated up to a 3.6fold increase in intrinsic dissolution rate compared with crystalline ENZ. These results identify aromaticity as a critical structural feature for coamorphous formation with ENZ and provide a rational basis for coformer selection for this drug and, by extension, other aromatic-rich, poorly soluble APIs. Abbreviations ENZ Enzalutamide ENZ-BEN Enzalutamide and benzoic acid ENZ-2AA Enzalutamide and 2-aminobenzoic acid ENZ-25H Enzalutamide and 2,5-dihydroxybenzoic acid ENZ-SAL Enzalutamide and salicylic acid (ENZ-SAL) T g Glass transition temperature PP Polypropylene SS Stainless steel mDSC Modulated differential scanning calorimetry ATR-FTIR Attenuated total reflection Fourier transform infrared spectroscopy IDR Intrinsic dissolution rate 1. Introduction With the increasing use of high-throughput screening technologies and computational chemistry, the number of newly discovered active pharmaceutical ingredients (APIs) increased. However, many APIs today exhibit poor aqueous solubility due to their increased molecular size and lipophilicity. Approximately 40 % of marketed drugs and 90 % of drugs in development face solubility-related challenges, leading to low bioavailability and limited therapeutic efficiency when administered orally.(Lipinski, 2002) To address these limitations, advanced drug delivery systems such as cocrystals(Chatziadi et al., 2022), micro-emulsions(Spernath and Aserin, 2006), nanocrystals (Gigliobianco et al., 2018), amorphous dispersions(Vasconcelos et al., 2016), etc. have been developed. However, these methods often involve complex formulation processes, scalability issues, or instability under physiological conditions.(Abaszadeh et al., 2023; Chavan et al., 2018; * Corresponding authors. E-mail addresses: [email protected] (A. Chatziadi), [email protected] (M. ˇ So´ oˇ s). Contents lists available at ScienceDirect European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps https://doi.org/10.1016/j.ejps.2025.107281 Received 28 April 2025; Received in revised form 7 September 2025; Accepted 16 September 2025 European Journal of Pharmaceutical Sciences 214 (2025) 107281 Available online 19 September 2025 0928-0987/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). LaFountaine et al., 2016) One of the most promising approaches in the development of drugdelivery systems to improve the solubility of the drug is amorphization.(Kim et al., 2021) Nevertheless, the use of single component amorphous drugs is limited by their tendency to recrystallize because of their thermodynamical instability. For better stabilization, polymers or mesoporous silica particles have been used as stabilizers.(Laitinen et al., 2013) The increased physical stability of these systems is explained by the enhanced glass transition temperature of the amorphous drug (T g ). However, these delivery systems have several drawbacks, including limited miscibility, increased dose volume, or hygroscopicity of the polymers that might potentially lead to recrystallization. Coamorphous systems, binary amorphous mixtures of an API and a small molecule coformer, have emerged as a promising formulation approach to improve the solubility of poorly water-soluble drugs. (Dengale et al., 2016a) Unlike polymer-based amorphous solid dispersions, coamorphous systems often require lower excipient load and exhibit improved physical stability when molecular interactions between the components hinder crystallization. Successful coamorphization typically depends on molecular-level miscibility and stabilization through hydrogen bonding, salt formation, or π – π interactions. Coamorphous systems exhibit higher stability together with an enhanced dissolution rate compared to individual compounds.(Dengale et al., 2016b) Enzalutamide (ENZ) (Fig. 1) is a non-steroidal antiandrogen drug used for the treatment of metastatic castration resistant prostate cancer. (Hoffman-Censits and Kelly, 2013) However, its crystalline form exhibits low aqueous solubility which in turn results in limited bioavailability via oral administration. To overcome this problem, various methods have been used including use of polymers and cyclodextrins, (Volkova et al., 2021) selfnanoemulsifying delivery systems,(Lee et al., 2024) amorphous solid dispersion,(Wilson et al., 2020) cocrystallization, nanocrystals(Guo et al., 2022) and graphene oxide nanoparticles. (Jiang et al., 2020) Amorphization has also been used; however, it has been shown that the amorphous phase is unstable and tends to recrystallize.(Romanov´ a et al., 2018a) ENZ, which lacks strong hydrogen bonding interactions in its crystal structure,(Maini et al., 2018) is an interesting case for coamorphous formulation. In a previous study, the formation of a coamorphous system of ENZ with saccharin is reported, (Prashanth et al., 2022a) in which it was suggested that the ability of ENZ to make eutectic with this molecule might be the reason to form coamorphous system. Still, the criteria driving its coamorphous behaviour remain unclear, and broader coamorphization with more molecules is needed to understand the mechanism of formation. In this study, we conducted a systematic screening of small organic acids and amino acids as potential coformers for developing coamorphous systems with ENZ. Our investigation revealed a prominent pattern; successful coamorphization occurred with coformers containing a benzene ring, including benzoic acid (BEN), salicylic acid (SAL), 2aminobenzoic acid (2AA), 2,5-dihydroxybenzoic acid (25H), L-phenylalanine (PHE), and L-tryptophan (TRY). To understand the role of benzene ring, we analyzed the crystal structure of ENZ, and we performed IR analysis. Furthermore, we investigated further properties like thermal stability, Room temperature (RT) stability and intrinsic dissolution rate. 2. Experimental section 2.1. Materials For the screening and dissolution experiments, the following chemicals were used: benzoic acid, salicylic acid, 2-aminobenzoic acid, 2,5dihydroxybenzoic acid, succinic acid, glutaric acid, pimelic acid, adipic acid, L-phenylalanine, L-tryptophan, L-methionine, L-leucine, Lproline, L-valine. From amino-acids only non-polar ones were selected, because it has been shown that they have the highest probability among other drugs to form co-amorphous systems.(Kasten et al., 2019) These were obtained from Merck. ENZ form R1 was kindly provided by Zentiva, k.s. Abbreviations and melting temperatures of the chemicals are presented in Table 1. All melting point data were retrieved from the PubChem Compound Database (https://pubchem.ncbi.nlm.nih.gov/) on June 18, 2025. 2.2. Preparation and characterization of the coamorphous systems 2.2.1. Visualization of the crystal structure of ENZ and calculations of interaction energies Calculation of interaction energies and energy frameworks was done using the software CrystalExplorer version 21.5.(Mackenzie et al., 2017) Molecular wave functions were obtained using the build-in Tonto utility at the “accurate” setting using the B3LYP/6-31G(d,p) level of theory. For the visualization of crystal structure of ENZ, the software Mercury (Macrae et al., 2008) was used. 2.2.2. Amorphous screening milling experiments A Retsch mm 400 mixer mill was used to prepare amorphous ENZ. Approximately 100 mg of crystalline ENZ were placed in polypropylene (PP) jars (2 ml Eppendorf tubes) together with two 5 mm stainless steel balls. The jars were then immediately closed. The oscillation frequency was 30 Hz. The milling period ranged from 30 to 90 min. 2.2.3. Coamorphous screening milling experiments Screening milling experiments were also performed with a Retsch mm 400 mixer mill. In a typical experiment, 100 mg of ENZ and the corresponding coformer either 1:1 or 2:1 ratio, were added to a 5 ml PP jar together with two 5 mm stainless steel balls and the jars were immediately closed. The oscillation frequency was 30 Hz, and the time of each experiment was from 90 to 120 min. Fig. 1. Molecular structure of Enzalutamide. Table 1 Chemicals that were used for this study, their abbreviation that is used in the text and their corresponding melting temperatures. Chemical Abbreviation Approx. Melting Temperature (◦C) Enzalutamide ENZ 200 Benzoic acid BEN 121 Salicylic acid SAL 158 2-aminobenzoic acid 2AA 146 2,5-dihydroxybenzoic acid 25H 204 Succinic acid SUC 184 Glutaric acid GLU 95 Pimelic acid PIM 103 Adipic acid ADP 152 L-phenylalanine PHE 280 L-tryptophan TRY 290 L-methionine MTH 281 L-leucine LEU 286 L-proline PRO 220 L-valine VAL 298 A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 2 2.2.4. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) Measurements were performed on a Nicolet 6700 Fourier transform infrared spectrometer (ThermoFisher Scientific) equipped with a deuterated triglycine sulphate detector and a ZnSe attenuated total reflection (ATR) accessory. The measuring range was from 4000 cm −1 to 650 cm −1 with a spectral resolution of 4 cm −1 . Data were obtained at 32 accumulations of the measured spectra. 2.2.5. Modulated differential scanning calorimetry (MDSC) Modulated DSC measurements were performed on a DSC3+(Mettler Toledo). Approximately 5 mg of the sample were weighed in an aluminum pan (volume 40 μ l) and covered. Measurements were carried out at a constant nitrogen flow rate of 50 ml/min and in modulated temperature mode. The samples were heated from 25◦C to 225◦C at a heating rate of 2◦C/min, an amplitude of 0.2120◦C and a period of 40 s. 2.2.6. Time-dependent stability studies The physical stability of the prepared coamorphous systems was evaluated under ambient conditions (25 ±2◦C, 40-60 % relative humidity) for extended periods. The samples were analysed by XRPD. 2.2.7. Determination of the intrinsic dissolution rate (IDR) of ENZ The absorbance of ENZ was measured using an Agilent Cary 60 UVVIS spectrophotometer. The measurements were carried out using an optical stainless-steel probe with a path length of 20 mm. The wavelength range was set from 190 to 600 nm with a scan speed of 600 nm/ min. The determination was performed at an absorption maximum of 280 nm. The drug concentration was calculated from a freshly prepared calibration curve. All measurements were made in triplicates. All samples were weighed in a small aluminum stub with an orifice ring diameter of 13 mm. The powder was pressed using an IR press at 5 kN for 60 seconds. An aluminum plate was placed on the wall of the measurement cell inside the dissolution medium. Concentration measurements were performed using ultrapure grade deionized water at a pH of 6.5, containing a small pre-dissolved amount of ENZ at a concentration of 1.0443 μ g/mL. Deionized water was used as the dissolution medium to eliminate the influence of buffer salts and pH on drug–coformer interactions, thereby enabling direct comparison of the intrinsic dissolution behavior of the different solid forms under neutral, unbuffered conditions. The solution was heated to 37◦C and stirred at 200 RPM. These conditions were kept constant throughout the experiment. The absorbance of the molecularly dissolved drug was monitored using a UVVIS probe every minute for a period of 240 min. The IDR was determined by calculating the linear slope between time and the concentration of the dissolved form of the initial 30 min of the dissolution process, divided by the surface area of the powder. The first five points were excluded from the calculation due to their typical reflection of dissolution by the free powder during disc preparation. The IDR measurements were not performed for coamorphous systems with BEN and TRY, due to overlapping absorbance at the region of interest. 3. Results and discussion 3.1. Insights into the crystal structure of ENZ To gain insight into the forces stabilizing the crystalline form of ENZ, its crystal structure was analyzed. As shown in Fig. 2, the crystal structure of ENZ reveals a molecular packing arrangement predominantly stabilized by π – π stacking interactions between the aromatic rings of adjacent molecules, while there is absence of significant hydrogen bonding networks. Furthermore, after calculation of the interaction energies between the molecules we can see that specifically in ENZ, π interactions are not so weak, with the strongest being -77.8 kJ-mol and the second strongest -37.8 kJ-mol (Fig. SI 1). The presence of these interactions in the crystalline lattice suggests that disruption of π – π stacking may be a key factor in enabling the transition to amorphous or coamorphous states. The impact of these structural changes is explored in the following sections through PXRD and IR spectroscopy. 3.2. Milling experiments for amorphization/coamorphization of Enzalutamide 3.2.1. Amorphization of enzalutamide Prior to developing coamorphous systems, it is essential to reveal the feasibility and conditions for amorphization of ENZ alone, as this serves as a reference point for evaluating the potential benefits of incorporating coformers. The transformation of crystalline ENZ to its amorphous form was investigated using XRPD. Fig. 3 illustrates the XRPD patterns of ENZ during the amorphization process at different time points (30, 45, 60, and 90 min). The diffractogram of the initial crystalline ENZ displays characteristic sharp diffraction peaks, indicating a well-ordered crystalline structure. Remarkably, after just 30 min of milling, the XRPD pattern shows a complete transformation to the amorphous state, as evidenced by the disappearance of all characteristic crystalline peaks (i. e., 9.8◦, 13.1◦, 19.7◦, 21.3◦, 22.8◦, 26.4◦) and the emergence of a broad halo pattern typical of amorphous materials. The subsequent patterns at 45, 60, and 90 min confirm the maintenance of the amorphous state, with all samples exhibiting similar broad halo patterns across the measured 2θ range (5-40◦). The ease of amorphization of ENZ suggests favourable thermodynamic and kinetic properties for the development of coamorphous formulations with selected coformers. 3.2.2. Screening of coformers for coamorphous formation with enzalutamide A systematic screening approach was employed to identify suitable coformers for coamorphous forms with ENZ. Initial screening was conducted using a 1:1 molar ratio of ENZ to potential coformers, followed Fig. 2. Illustration of the most dominant aromatic π – π interactions between molecules in Enzalutamide. Fig. 3. XRD pattern of crystalline Enzalutamide, with the characteristic reflections from the left to the right: (1) 9.8◦, (2) 13.1◦, (3) 19.7◦, (4) 21.3◦, (5) 22.8◦, (6) 26.4◦and XRPD diffractograms of ENZ after milling for a period of a) 30min, b) 45 min, c) 60 min, d) 90 min. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 3 by optimization at a 2:1 ratio for selected compounds. A diverse range of molecules was evaluated, including aromatic and non-aromatic carboxylic acids and amino acids with varying structural features. The results of the successful coamorphous systems are presented in Fig. 4. Carboxylic acids as coformers We first investigated a diverse range of carboxylic acids as potential coformers, including both aromatic and non-aromatic structures with varying functional groups. Table S1 summarizes the results of carboxylic acid screening. A clear structure-dependent pattern emerged from the carboxylic acid screening. Specifically in Fig.s SI 2 and SI 3, we can see that only partial amorphization was achieved for all the systems for ratio 1:1. Thus, extended milling times (up to 120 min) were investigated to determine whether complete amorphization could be achieved. Nevertheless, as we can see in Fig.s SI 4 and SI 5, complete amorphization was not attained at the ratio 1:1, suggesting that the limitation was not kinetic but rather related to unsatisfied molecular interactions at this specific stoichiometry. When the molar ratio was adjusted to 2:1 (ENZ-coformer), all four systems with aromatic carboxylic acids achieved complete amorphization, as confirmed by PXRD patterns showing only the characteristic amorphous halo with no detectable crystalline peaks (Fig. 5). Interestingly, none of the non-aromatic carboxylic acids formed coamorphous systems, and in addition, in most cases, the peaks of the coformers, but also the peaks of ENZ, were even more intensive at 2:1 ratio (Fig. SI 6), confirming that the absence of an aromatic ring was the critical limiting factor. Amino acids as coformers To further investigate the role of aromatic structures in coamorphous formation, we extended our screening to include several amino acids with different side chain properties. Table SI 2 presents the results of amino acid screening which further confirmed the importance of aromatic structures. As shown in Fig. 6, PHE and TRY, both containing aromatic side chains, formed completely amorphous systems with ENZ even at the initial 1:1 ratio. Notably, unlike the aromatic carboxylic acids, these amino acids did not require ratio optimization to achieve complete amorphization, suggesting potentially more favourable interactions with ENZ. In contrast, amino acids without aromatic groups (MTH, LEU, PRO, VAL) failed to form coamorphous systems with ENZ, even after prolonging the milling time or changing the ratio to 2:1 (Fig.s SI 7 and SI 8). Structure-Dependency in Coamorphous Formation The comprehensive screening of both carboxylic acids and amino acids revealed a structure-dependent pattern in coamorphous formation with ENZ. Only coformers containing a benzene ring successfully formed coamorphous systems, while all non-aromatic compounds failed despite varied functional groups and hydrogen bonding capabilities. Furthermore, the screening results highlighted that even among aromatic coformers, there were differences in coamorphous-forming ability; aromatic amino acids achieved complete amorphization at 1:1 ratio, while aromatic carboxylic acids required a 2:1 ratio for complete amorphization. This distinction suggests that the specific positioning and nature of functional groups attached to the aromatic ring influence the interactions with ENZ and the resulting amorphization behaviour. This clear correlation between molecular structure and coamorphous formation suggests that the benzene rings play a crucial role in stabilizing the amorphous state with ENZ, either through some π interactions or by disrupting the crystallization process, or by shape complementarity. To understand better this phenomenon, we employed more characterization techniques. 3.3. IR analysis To gain additional insights into the interactions within the coamorphous systems of ENZ, we used vibrational spectroscopy analysis. As we can see in Fig. 7, The FTIR spectrum of crystalline ENZ displays several characteristic absorption bands, which were assigned according to the literature.(Prashanth et al., 2022b) The strong band at ~1670 cm⁻¹ corresponds to the C=O stretching vibration of the amide carbonyl group. Peaks in the 1600–1500 cm⁻¹ region can be attributed to aromatic C=C stretching modes of the benzene rings, while the band at ~1250 cm⁻¹ is assigned to C–N stretching. Vibrations in the region 1150–1100 cm⁻¹ are consistent with C–F stretching of the fluoro-substituted aromatic ring. The fingerprint region below 1000 cm⁻¹ contains characteristic out-of-plane bending modes of the aromatic C–H bonds. Upon amorphization and co-amorphous formation, broadening and merging of the peaks is observed at the region of aromatic C=C stretching modes, indicating disruption of π – π stacking interactions. These spectral transformations provide evidence that amorphization induces comprehensive molecular rearrangement in ENZ, disrupting the uniform π - π intermolecular interactions of the crystalline state in favour of more variable, less directional interactions in the amorphous form. Analysis of the ENZ co-amorphous systems with different coformers revealed two general trends. For 2AA, 25H, and SAL (Fig.s SI 9–11), no significant shifts or new bands were detected compared to amorphous ENZ, suggesting the absence of strong, specific molecular interactions. These systems likely represent homogeneous dispersions where the coformers are molecularly mixed within the ENZ amorphous matrix. This molecular-level mixing can be driven by factors such as weak intermolecular forces, molecular sizes and shapes, complementary molecular structures, and thermodynamic compatibility, which in turn can promote efficient molecular interactions and the formation of a homogeneous phase. Similar findings have been made previously to explain the stabilization of co-amorphous mixtures.(L¨ obmann et al., 2012) In contrast, the ENZ-BEN system (Fig. 7) showed distinct spectral changes, particularly in the carbonyl stretching region (1650–1600 cm⁻¹). Compared to both crystalline benzoic acid and amorphous ENZ, a clear shift was observed, consistent with the formation of hydrogen bonding between the carboxylic acid group of BEN and the carbonyl of ENZ. We also carefully examined the aromatic skeletal vibration region (1600-1500 cm⁻¹) in the ENZ-benzoic acid co-amorphous spectrum. Compared to crystalline ENZ, ENZ-BEN show broadened bands in this region, reflecting loss of ordered π - π stacking. However, the ENZ-BEN spectrum does not show additional shifts or changes beyond those already observed for amorphous ENZ, suggesting that the principal additional interaction in this system is hydrogen bonding involving the carbonyl groups, rather than further modification of π - π stacking. This bond is the main additional stabilization. π - π interactions are weak and delocalized and amorphous spectrum is already broad, so we are not able to see the small shifts. Fig. 8 The ENZ-PHE coamorphous system (Fig. 9) exhibited a spectrum largely resembling amorphous ENZ, with only one slight shift observed in the 1580-1600 cm⁻¹ region corresponding to the carboxylate asymmetric stretching of phenylalanine. This suggests that PHE, similarly to BEN, 25H and SAL, is likely dispersed within the amorphous ENZ matrix without forming strong specific interactions. Similarly, the ENZ-TRY coamorphous system displayed spectral Fig. 4. XPRD results from 90 min milling experiments and different ratio for successful coamorphous systems. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 4 features predominantly matching amorphous ENZ, with a distinctive alteration observed in the 750 cm⁻¹ region associated with indole ring vibrations of tryptophan. In this region, the characteristic sharp tryptophan peak appears to mask the two smaller peaks typically observed in amorphous ENZ, indicating potential π - π stacking interactions between TRY’s indole ring and ENZ’s aromatic moieties. The limited nature of spectral modifications in both coamorphous systems indicates that while specific functional group interactions exist, they do Fig. 5. XPRD results from 90 min milling experiments of ENZ with benzoic acid derivatives for ratio 1:1 and 2:1. We observe that the amorphization is complete in ratio 2:1. Fig. 6. XPRD results from 90 min milling experiments of ENZ with amino acids for ratio 1:1. We observe that for LEU, MTH, PRO and VAL there are remaining peaks of the coformers, suggesting extension of milling period. Fig. 7. Experimental IR spectra of crystalline and amorphous ENZ, crystalline BEN and coamorphous system ENZ-BEN. Fig. 8. Experimental IR spectra of crystalline and amorphous ENZ, crystalline PHE and coamorphous system ENZ-PHE. Fig. 9. Experimental IR spectra of crystalline and amorphous ENZ, crystalline PHE and coamorphous system ENZ-PHE. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 5 not induce comprehensive molecular rearrangements throughout the structures. We note that in this work, FTIR analysis was performed at selected drug-to-coformer ratios chosen based on literature precedent and prior evidence for stable co-amorphous formation. A broader stoichiometric screening, combined with multivariate FTIR data analysis, could provide deeper quantitative insight into how the extent and nature of molecular interactions vary with composition. This is planned for future studies to expand on the findings reported here. 3.4. Thermal analysis of ENZ and its coamorphous systems To investigate the thermal behaviour of the prepared coamorphous systems, we used mDSC to study the T g . Table 1 presents the results for amorphous ENZ and all our systems. Table 2 Amorphous ENZ exhibited a glass transition temperature of approximately 95◦C (Fig. SI 12). Notably, the recorded T g temperature of amorphous ENZ in our study is found to be different from those reported in related work. For instance, amorphous ENZ prepared by lyophilization(Prashanth et al., 2022c) exhibits T g at 48◦C, while in another study, amorphous ENZ prepared by hot melt extrusion (Romanov´ a et al., 2018a) and thermal evaporation exhibited T g at 85◦C and 72◦C, respectively. This highlights the significant influence of preparation methods on the T g of amorphous forms. In our case, it is evident that ball milling is a superior method to prepare amorphous form since ENZ obtained from ball milling has the highest T g , indicating that probably also exhibits higher stability. Differences in Tg values of amorphous ENZ across studies can indeed arise from the use of different preparation methods, as each method can introduce subtle variations in the solid-state properties. For example, ball milling, used in our study, typically yields high-density amorphous solids due to mechanical compaction and intimate mixing, potentially resulting in higher Tg compared to other techniques. Moreover, several additional factors can contribute to Tg variability such as, residual moisture, which acts as a plasticizer and lowers T, existence of polyamorphism, where different amorphous forms of the same compound can have distinct thermodynamic properties, including Tg or chemical degradation during processing, which may lead to the presence of minor impurities or degradation products that affect the glass transition behaviour.(Musumeci et al., 2025) All successful coamorphous systems exhibited a single T g , confirming the formation of homogeneous single-phase systems. The coamorphous systems formed with BEN, SAL, and 2AA showed lower T g values (6070◦C) compared to amorphous ENZ alone. In contrast, the ENZ-25H coamorphous system exhibited a slightly higher T g (73◦C) than the other benzoic acid derivatives, though still below that of pure amorphous ENZ. Additionally, in all thermographs (Fig. SI 13) we observe an extra endothermic peak that does not correspond either to the melting point of ENZ or to the corresponding coformer in each case. Since in earlier studies the ability of ENZ and saccharin to form eutectic systems was reported,(Prashanth et al., 2022c) we assumed that this would also be the case for our systems. To confirm this, we performed DSC measurements of physical mixtures of our systems in the same ratio as our coamorphous forms. Indeed, it was confirmed that these extra peaks that we observe in our systems correspond to their eutectics (Fig. SI 16). Their ability to form eutectics might be an extra reason for the formation of coamorphous with these specific compounds, indicating high miscibility. In most cases, the ratio didn’t match precisely the eutectic point, so we also observe peaks that correspond to the melting temperature of ENZ and the coformers. But it is interesting to note that in the case of ENZ-25H, in the DSC of the mixture we observe only one peak, indicating this ratio corresponds to the eutectic composition. The aromatic amino acids demonstrated different thermal behaviour compared to the benzoic acid derivatives. The ENZ-PHE coamorphous system showed a T g of approximately 90◦C, which is close to that of amorphous ENZ alone. Most notably, the ENZ-TRY coamorphous system demonstrated a T g (approximately 106◦C) that was higher than that of amorphous ENZ alone. 3.5. Time-dependent stability study The stability over time of an amorphous or coamorphous system is very important because if it recrystallizes it means that the advantages of higher solubility and dissolution over the pure crystalline form of the drug will be lost. For that reason, the physical stability of the prepared coamorphous systems was evaluated under ambient conditions (room temperature, 40-60 % relative humidity) for extended periods. Stability of Amorphous Enzalutamide As shown in Fig. 10, amorphous ENZ alone demonstrated remarkable physical stability, maintaining its amorphous character for up to 150 days of storage under ambient conditions. The XRPD patterns collected at various time points (0, 7, 30, 60, 90, and 150 days) consistently exhibited the characteristic amorphous halo pattern with no emergence of crystalline peaks. This inherent stability of amorphous ENZ provided a solid foundation for the development of coamorphous systems with enhanced physical properties. Stability of Aromatic Carboxylic Acid Coamorphous Systems The coamorphous systems prepared with aromatic carboxylic acids displayed varying stability over time (Fig. 11). The ENZ–BEN system remained amorphous for approximately 7 days. By 30 days, distinct reflections of ENZ (e.g., at ~2θ =13.8◦and 19.6◦; black stars) and BEN (e.g., at ~8◦and 16.3◦; red stars) became evident, confirming phase separation and recrystallization of both components (Fig. 11a). Similarly, the ENZ–2AA system showed an amorphous halo up to 7 days. At 30 days, sharp reflections reappeared at positions corresponding to ENZ and 2AA (~15.1◦and 23.2◦; red stars), indicating recrystallization of both components (Fig. 11b). After 60 days, the crystallinity intensified, with stronger reflections dominating the diffractogram. The ENZ–SAL system exhibited a slightly higher stability compared to BEN and 2AA, retaining a higher percentage of amorphous phase for 30 days. By 60 days, however, ENZ and SAL peaks (~10.9◦and 17.1◦; red stars) appeared (Fig. 11c). Table 2 Experiments Glass Transition Temperatures (T g ) for all the successful coamorphous systems. Amorphous System Glass transition Temperature (T g ) o C ENZ am. 95 ENZBEN 60 ENZ-SAL 65 ENZ2AA 64 ENZ25H 73 ENZPHE 95 ENZTRY 106 Fig. 10. Stability studies study over time for amorphous ENZ. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 6 In contrast, the ENZ–25H system demonstrated superior stability, maintaining a fully amorphous pattern up to 60 days. Only faint ENZ reflections (~13.8◦; black star) were detectable after 90–120 days, while no distinct 25H peaks were observed (Fig. 11d). This result highlights the stabilizing effect of 25H, consistent with its higher Tg and alignment with eutectic composition. Stability of Aromatic Amino Acid Coamorphous Systems The coamorphous systems with aromatic amino acids also showed delayed recrystallization compared with the carboxylic acid systems (Fig. 12). The ENZ–PHE system preserved its amorphous halo for 31 days. After 57 days, crystalline reflections characteristic of ENZ reappeared at ~2θ =13.1◦and 23◦(black stars, Fig. 12a). The ENZ–TRP system demonstrated similar stability. Up to 32 days, the amorphous halo remained intact. By 59 days, characteristic ENZ peaks were visible, along with a reflection attributable to TRP at ~2θ = 18.5◦(red star, Fig. 12b). These results indicate that both amino acid systems undergo recrystallization after nearly two months, but show significantly improved stability compared with the ENZ–BEN, ENZ–2AA, and ENZ–SAL systems. The higher stability of the amino acid coamorphous systems correlates with their elevated glass transition temperatures compared to the carboxylic acid systems. Correlation of stability with T g values Stability studies revealed an unexpected relationship between glass transition temperature and physical stability. While the observed stability generally followed the pattern predicted by T g values for most systems (with higher T g correlating with better stability), the ENZ-25H system presented an intriguing exception. Despite having a lower glass transition temperature (73◦C) than both ENZ-PHE (95◦C) and ENZ-TRY (106◦C) coamorphous systems, the ENZ-25H coamorphous system exhibited superior long-term physical stability, maintaining its Fig. 11. XRPD patterns of coamorphous ENZ–coformer systems during stability studies under ambient conditions. Representative crystalline peaks of ENZ are marked with black stars, while peaks corresponding to the respective coformers are marked with red stars. (a) ENZ–2AA system, (b) ENZ–SAL system, (c) ENZ–BEN system, and (d) ENZ–25H system. The reappearance of these peaks at later time points indicates recrystallization and phase separation. Fig. 12. XRPD patterns of coamorphous ENZ–amino acid systems during stability studies under ambient conditions (25 ±2◦C, 40–60 % RH). Representative crystalline peaks of ENZ are indicated with black stars, while peaks corresponding to the coformer are marked with red stars. (a) ENZ–PHE system and (b) ENZ–TRP system. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 7 amorphous character for at least 90 days with only minimal crystallization at 120 days. This contradiction highlights the complex interplay of factors beyond just T g that influence the physical stability of coamorphous systems. This can be explained by identifying that ENZ-25H uniquely forms at a ratio that corresponds precisely to its eutectic composition. This alignment with the eutectic point creates a thermodynamically favoured state characterized by minimized Gibbs free energy, optimal molecular packing, and reduced driving force for phase separation or crystallization. This optimal molecular arrangement at the eutectic composition effectively raises the kinetic barrier to crystallization despite the system’s intermediate T g . Our findings support previous research suggesting that matching the coamorphous system’s composition to its eutectic point is critical for maximizing physical stability,(Kissi et al., 2019) and demonstrates that while T g remains an important indicator, the relationship between composition, molecular interactions, and stability is more complex. To further support the importance of different ratio for stabilizing coamorphous systems, exhaustive ratio screening was not feasible here due to the milling-based preparation route, which does not consistently yield amorphous forms across all ratios. Future studies will therefore focus on complementary techniques (e.g., melt quenching) to systematically explore ratio-dependent effects 3.6. Intrinsic dissolution rate (IDR) of ENZ solid forms In this study, dissolution performance was assessed using the intrinsic dissolution rate (IDR) method, which provides a controlled comparison of the inherent release potential of different solid forms by eliminating variability in surface area, particle size, and dosage form geometry. This approach allowed us to focus specifically on the contribution of solid-state modifications—particularly the role of aromatic π – π interactions—to dissolution behavior. The IDR of crystalline and amorphous ENZ, and various coamorphous systems was investigated to evaluate the impact of coamorphization on drug dissolution behaviour. Fig. 13 presents the IDR values obtained for all systems under standardized conditions. Crystalline ENZ exhibited the lowest IDR value (0.45 μ g⋅cm⁻²⋅min⁻¹), consistent with its poor aqueous solubility. Conversion to the amorphous state resulted in a modest improvement, with amorphous ENZ showing an approximately 1.5-fold increase in IDR (0.65 μ g⋅cm⁻²⋅min⁻¹). This enhancement can be attributed to the higher energy state and absence of a crystal lattice in the amorphous form. Remarkably, all coamorphous systems demonstrated substantially higher IDR values compared to both crystalline and amorphous ENZ alone. The coamorphous system with PHE showed an IDR of 0.80 μ g⋅cm⁻²⋅min⁻¹, representing a 1.8-fold improvement over crystalline ENZ. The 2AA coamorphous system exhibited a further enhancement with an IDR of 1.05 μ g⋅cm⁻²⋅min⁻¹. More significant improvements were observed with SAL and 25H coamorphous systems, which achieved IDR values of 1.38 and 1.62 μ g⋅cm⁻²⋅min⁻¹, respectively, corresponding to 3.1-fold and 3.6-fold increases compared to crystalline ENZ. The dissolution enhancement followed the order: crystalline ENZ < amorphous ENZ <ENZ-PHE <ENZ-2AA <ENZ-SAL <ENZ-25H. This trend suggests that the specific structural features of the coformers influence not only the formation of coamorphous systems but also their dissolution behaviour. Notably, the coformers with hydroxyl (-OH) substituents on the benzene ring (SAL and 25H) provided the most significant dissolution improvements. The superior dissolution performance of the 25H coamorphous system is particularly interesting when considered alongside its thermal properties. Despite having a higher T g than other carboxylic acid coamorphous systems, it demonstrated the highest IDR. This suggests that while the 2,5-dihydroxy pattern might provide π - π interactions that stabilize the amorphous state (resulting in higher T g ), these interactions do not slow down drug release upon exposure to dissolution media. In fact, the hydroxyl groups may enhance wettability and increase the hydrophilicity of the coamorphous matrix, facilitating more rapid dissolution despite the greater physical stability. These findings demonstrate that coamorphization with aromatic coformers not only enables the formation of stable amorphous systems but also substantially enhances the dissolution rate of ENZ. The ability to simultaneously improve both physical stability and dissolution behaviour makes these coamorphous systems particularly promising for addressing the biopharmaceutical limitations of ENZ. While IDR is well suited for early-stage screening and mechanistic studies, we acknowledge that further evaluation under formulationrelevant conditions is needed to fully translate these findings into product development. In particular, assessing supersaturation generation and maintenance in biorelevant media, as well as conventional dissolution testing for prototype dosage forms, will be the focus of our subsequent work. 4. Conclusions This study provides a mechanistic understanding of coamorphous system formation in Enzalutamide (ENZ), highlighting the critical role of aromaticity and possible π – π interactions in coformer selection. Through systematic screening, we observed that only coformers containing aromatic rings could form fully amorphous binary systems with ENZ, while linear or non-aromatic coformers failed to induce complete amorphization. Crystal structure analysis revealed that ENZ is stabilized in its crystalline form primarily through π – π stacking, rather than hydrogen bonding. The formation of coamorphous systems with aromatic coformers is attributed not to the creation of new strong interactions, but rather to the stabilization of its amorphous form, in which it is shown that there is weakening or partial disruption of the π – π networks and improved molecular mixing. Additionally, the geometric compatibility between the benzene rings of the coformer and the aromatic moiety of ENZ promotes efficient molecular packing in the amorphous phase, preventing phase separation and ensuring structural stability. In contrast, linear coformers lack the aromaticity required to establish such interactions with ENZ. As a result, they fail to disrupt the crystalline π - π stacking effectively and cannot provide the necessary stabilization for the amorphous form. The absence of π - π interactions with linear coformers also probably leads to unfavourable packing in the amorphous state, introducing voids or irregularities that likely result in phase instability or separation. Thermal analysis confirmed the formation of single-phase amorphous systems with elevated glass transition temperatures, while storage ENZcr. ENZam. PHE 2AA SAL 25H 0.0 0.4 0.8 1.2 1.6 2.0 (/etaRnoitulossiDcisnirtnI -2 min -1 ) Pg cm Fig. 13. Comparison of the IDRs of the crystal, amorphous, and co-amorphous form of ENZ with respect to the IDR of the crystal form. A. Chatziadi et al. European Journal of Pharmaceutical Sciences 214 (2025) 107281 8 stability studies demonstrated that the ENZ-2,5-dihydroxybenzoic acid (ENZ–25H) system, corresponding to the eutectic composition, remained amorphous for up to four months. This system also exhibited a 3.6-fold increase in intrinsic dissolution rate compared to crystalline ENZ, probably due to the number of OH groups, which enhance the wettability and increase the hydrophilicity of the coamorphous matrix. This study highlights key structural features driving coamorphous formation and may guide the design of coamorphous systems for other poorly soluble, aromatic-rich APIs, broadening the applicability of this strategy beyond ENZ. Supporting Information Further details regarding interaction energies, results of milling experiments, experimental IR spectra for the rest of the systems, DSC of coamorphous systems and DSC of physical mixtures, and concentration of enzalutamide obtained during the IDR measurements. Notes The authors declare no competing financial interest. CRediT authorship contribution statement Argyro Chatziadi: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Conceptualization. Kateˇ rina Neubergerov´ a: Methodology, Investigation. Venkata Krishna Rao Balaga: Writing – review & editing, Methodology, Investigation. Dan Trunov: Writing – review & editing, Methodology, Investigation. Miroslav ˇ So´ oˇ s: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Conceptualization. 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