Overcoming itraconazole limitations via proline cocrystals: improved solubility, dissolution, and antifungal efficacy

Abstract

Crystal engineering plays a crucial role in the efficient design of pharmaceutical cocrystals, hinging on a deep understanding of intermolecular interactions that influence crystal packing. By leveraging these interactions, researchers can create innovative solid forms with customized physical and chemical properties. This versatile approach is especially valuable for tackling the solubility and bioavailability issues commonly associated with poorly soluble drugs. In this study, we focused on synthesizing cocrystals of itraconazole (ITZ) and proline via the kneading technique, an eco-friendly crystal engineering technique. Our work included comprehensive solid-state characterization, stability assessments, and in vitro efficacy evaluations. We specifically examined how these cocrystals performed in terms of dissolution, solubility, and antifungal activity. Our solid-state characterization results highlighted the formation of significant intermolecular interactions within the cocrystals, particularly hydrogen bonds between ITZ and the functional groups of proline. Notably, the ITZ-proline cocrystals demonstrated superior solubility and faster dissolution rates compared to pure ITZ, alongside increased crystallinity in the newly formed solid state. Additionally, the cocrystals exhibited enhanced antifungal activity and stability. Consequently, our findings have indicated that forming ITZ cocrystals with proline significantly enhances the solubility as well as stability of ITZ, thereby offering a promising antifungal strategy to combat fungal infections.

Introduction

In recent years, the field of crystal engineering for active pharmaceutical ingredients (APIs) has gained significant attention among formulation specialists. The pharmaceutical industry often favors crystalline forms of APIs due to their inherent thermodynamic stability. However, these crystalline APIs can exist in multiple polymorphic forms, which can lead to challenges such as poor solubility and inadequate dissolution rates. This is particularly problematic for compounds that are not easily soluble in water, ultimately limiting their oral bioavailability. The challenge of low solubility in new chemical entities (NCEs) has become a significant hurdle in drug discovery and development. Compounds with poor solubility complicate both in vitro and in vivo testing, creating a substantial obstacle in the drug development process. Research indicates that around 75% of drug candidates in development face solubility issues, particularly those classified as BCS (biopharmaceutical classification system) classes II and IV. These classes are characterized by their low solubility and permeability, which are critical for effective oral absorption. Cocrystallization offers a promising strategy to enhance the physical and mechanical properties of drugs, improving the overall performance of the API without compromising its pharmacological or intrinsic properties. This method provides an alternative means to address solubility challenges and advance drug formulation ^{1, 2)} .

Itraconazole (ITZ) is a triazole antifungal agent characterized as a weak base, with a molecular weight of 705.6 g/mol and pKa values of 2 and 3.7, alongside a log P of 6.2. It is known for its extremely low solubility in water, registering just 5 μg/ml at pH 1 and 1 μg/ml at pH 7, while exhibiting high permeability. Classified as a BCS class II compound, ITZ is practically insoluble in aqueous solutions. This medication is commonly used to treat various fungal infections, including histoplasmosis, blastomycosis, cryptococcal meningitis, and aspergillosis. Generally, ITZ is well tolerated at doses below 200 mg per day, although its oral absorption can be inconsistent. The drug has an elimination half-life ranging from 30 to 64 hours, with an absolute oral bioavailability of approximately 55%. Additionally, it has a high binding rate, around 99.8% ^{2, 3)} . The literature reveals various approaches to forming cocrystals of ITZ using different cocrystal formers, also called as coformers ^{4, 5)} . Though research on ITZ cocrystals has progressed, only a few studies have focused on their stability, a crucial factor for their practical application. Stability remains an under-investigated area, limiting the understanding of how these cocrystals perform over time. Furthermore, commonly used preparation methods pose significant challenges. These methods are not only expensive but also present issues with scalability, hindering their use in large-scale production. From an industrial perspective, the kneading method for synthesizing cocrystals is advantageous due to its reduced solvent requirements, thereby making it both cost-effective and aligned with green chemistry principles ⁶⁾ .

In the present investigation, proline was used as a coformer after virtual screening, as it stands out as a promising candidate for use as a cocrystal former. It features a zwitterionic a-ammonium-carboxylate synthon that is characteristic of all natural amino acids, enhancing intermolecular interactions through enthalpic contributions. Unlike many other amino acids, proline possesses a rigid structure due to its unique five-membered ring “lateral chain,” which is relatively uncommon among amino acids. This rigidity provides an entropic advantage in the formation of pharmaceutical cocrystals compared to more flexible coformers. Additionally, proline’s high solubility in water further enhances its suitability as a cocrystal former ⁷⁾ . This investigation focuses on modifying the physicochemical properties of ITZ by utilizing proline as a coformer by employing the kneading technique to enhance the overall characteristics of ITZ.

Materials and Methods

ITZ was generously provided as a sample by Glenmark Research Centre, located in Navi Mumbai, India. Proline was obtained from Loba Chemie, also based in Mumbai. All other chemicals used in this study were of analytical grade and sourced from Loba Chemie as well.

Virtual screening of cocrystal formers

Molecular docking serves as a powerful method for structure-based drug discovery using computational techniques. This approach estimates how likely one molecule, the API, will bind and orient itself with another molecule, known as the coformer, to create a new complex. The binding affinity between the two molecules can be evaluated through various scoring functions. In this study, nine coformers were screened for cocrystallization utilizing PyRx software ⁸⁾ .

Preparation of ITZ-proline cocrystals by the kneading technique

In this process, an equimolar quantity of ITZ (705.06 mg) and proline (115.13 mg) was measured out in a mortar at a 1 : 1 molar ratio. Ethanol was added to this mixture, and it was triturated with a pestle until a damp mass was formed. This mixture was then allowed to dry at room temperature and passed through a 60-mesh sieve before being stored in a desiccator for later use. Additionally, a simple physical mixture of the ITZ and proline was also prepared ⁹⁾ .

Solid-state characterization

The characterization of pure ITZ, proline, their physical mixture, and the formulated product was carried out using Fourier transform infrared (FTIR) spectroscopy on a Shimadzu FTIR 8300S instrument from Tokyo, Japan. Differential Scanning Calorimetry (DSC) was conducted with a DSC-60A calorimeter (Shimadzu, Tokyo, Japan), where samples were heated in hermetically sealed aluminum pans under a nitrogen atmosphere (30 ml/min) at a rate of 100 °C/min, ranging from 50°C to 300°C. For assessing crystallinity, an X-ray diffractometer (Bruker AXS Analytical Instrument, D2-PHASER, Germany) was employed, featuring a LYNXEYE™ detector and utilizing filtered K radiation from a Cu tube at 10 mA and 30 kV. Proton NMR spectra were obtained using a Bruker Avance 400 FT NMR spectrometer from SAIF, calibrated to TMS as an internal reference, and recorded at a temperature of 25°C with chloroform (CDCl3) as the solvent. Raman spectra for both the pure drug and the formulations were collected in the range of 0-5000 cm ⁻¹ on a Bruker Multi RAM Raman spectrometer, equipped with an Nd : YAG laser at an excitation wavelength of 1064 nm and a resolution of 4 cm⁻¹. These Raman spectra were recorded both before and after conducting stability studies, which were performed under storage conditions of 75% relative humidity and 40°C for a duration of three months ^{10, 11)} .

Saturation solubility of the drug and prepared cocrystals in SGF (pH 1.2)

Solubility analysis was conducted following the method outlined by Higuchi and Connors in simulated gastric fluid (SGF) at pH 1.2. The resulting solution was filtered using a 0.45 µm Whatman filter paper, and appropriate dilutions were made with SGF at the same pH. The quantity of solubilized drug was then assessed using UV spectroscopy at a wavelength of 254 nm ¹²⁾ .

Determination of percentage yield and drug content

To assess the drug content, 10 mg of the prepared cocrystals was triturated with simulated gastric fluid (SGF) at pH 1.2, and the mixture was brought to a total volume of 100 ml. The solution was then filtered through a 0.45 µm membrane. Following appropriate dilution with SGF (pH 1.2), the sample was analyzed spectrophotometrically at a wavelength of 254 nm ¹³⁾ .

In vitro dissolution studies

In vitro dissolution studies of ITZ and the prepared cocrystals were performed in triplicate using an eight-station USP type II dissolution apparatus (Veego VDA-14D). Samples equivalent to 200 mg of ITZ were precisely measured. The dissolution was carried out in 900 ml of simulated gastric fluid (SGF) (pH 1.2) at a temperature of 37±0.5°C and a stirring speed of 100 rpm. At regular intervals, samples were withdrawn, and fresh dissolution medium was added to maintain sink conditions. The samples were then filtered, diluted, and analyzed at 254 nm using a UV-Vis spectrophotometer (SHIMADZU CORP. 80906 UV-1900) ¹⁴⁾ .

Stability studies

Stability studies of the formulation were conducted under conditions of 75% relative humidity and 40°C for a duration of three months using an SC-6 PLUS REMI Stability Chamber. Samples were taken each month to evaluate drug content, as well as to observe any changes in color, odor, and overall appearance. After three months, the formulations were further characterized using Raman spectroscopy ¹⁵⁾ .

In vitro antifungal activity

The antifungal efficacy of ITZ and its cocrystals was evaluated against Aspergillus niger and Candida albicans using the well plate assay. The compounds were dissolved in DMSO and introduced into wells on Sabouraud Dextrose agar plates that had been inoculated with standardized fungal suspensions (10^7 CFU/ml). Concentrations of 1, 3, and 5 µg/mL were tested, along with a standard ITZ solution. Control wells contained only DMSO. The plates were incubated at 34°C, and the inhibition zones were measured after 48 hours ¹⁶⁾ .

Statistical analysis :

All experiments were performed in triplicate, and results were presented as mean ± SD. One-way ANOVA followed by Tukey’s post hoc test was used to compare the three formulations.

Results and Discussion

Virtual screening

Proline was evaluated among the nine coformers for its interaction potential, compatibility, and docking scores to confirm the formation of cocrystals with ITZ. The results of the virtual screening are summarized in Table 1. Based upon the docking score, proline was selected as a coformer, as the score indicated higher binding energy.

Table 1. Results of possible structures and docking score of ITZ with different cocrystal coformers.

FTIR spectroscopy

The infrared spectroscopy analysis reveals distinct characteristics in the spectra of the compounds examined, offering insights into molecular interactions and structural changes during the formation of cocrystals, as illustrated in Figure 1. For ITZ, absorption bands in the range of 2,800 to 3,200 cm ⁻¹ are associated with alkane, aromatic CH, and amine groups. In the aromatic NH region, proline shows notable absorption peaks at 3,365 cm ⁻¹. Interestingly, the cocrystals exhibit noticeable shifts in the aromatic CH region compared to the physical mixture. In the carbonyl (C=O) region, ITZ presents an absorption peak at 1,697 cm ⁻¹, while proline displays an absorption at 1,649 cm ⁻¹. A new absorption band appears at 711 cm ⁻¹ in the cocrystals, indicating unique molecular configurations. Moreover, a band at 3,140 cm ⁻¹, corresponding to the O-H of the carboxylic group in proline, shifts to 3,098 cm ⁻¹ in the cocrystals, suggesting strong hydrogen bonding. Overall, these spectroscopic findings provide valuable insights into the intricate molecular interactions and structural changes that occur during cocrystal formation ^{17, 18)} .

Fig. 1.

FTIR spectra of pure ITZ, proline, physical mixture, and cocrystals.

DSC analysis

Pure ITZ exhibits a melting point of 180°C, consistent with its documented melting behavior, showing no polymorphic transitions during the heating process. Proline, the other component studied, has a melting endotherm at 207.7°C. When ITZ and proline are combined in a physical mixture, the melting point drops to 167.1°C. This reduction suggests a possible interaction between the two compounds, leading to a lower melting point than that of the pure substances. The cocrystals formed from ITZ and proline display an even lower melting point of 158.5°C, as shown in Figure 2. This significant decrease indicates the formation of new solid-state structures with altered thermal properties. In the kneading method, the reduced melting point can be attributed to the breaking and reformation of bonds. The lower melting point of the cocrystals implies that the interactions within the cocrystal lattice create a reduced energy barrier for melting compared to the individual components or their physical mixture. In summary, the DSC data indicate a single endothermic event with a melting point lower than that of the original components, confirming the formation of cocrystals ^{19- 21)} .

Fig. 2.

Comparative DSC Analysis of ITZ, proline, physical Mixture (ITZ + Proline), and cocrystals.

X-ray diffractometry (XRD) analysis

The XRD patterns of both the pure drug and the cocrystals are presented in Figure 3. The XRD analysis of pure ITZ reveals prominent Bragg peaks at 10.71°, 14.38°, 17.40°, 20.31°, 23.40°, 25.22°, and 27.00° (2θ), with peak intensities of 538, 1023, 1537, 1947, 1278, 1102, and 899, respectively, indicating its crystalline structure. Proline displays characteristic crystalline peaks at 2θ values of 8.59°, 14.62°, 17.14°, 19.05°, 22.40°, 25.10°, 26.32°, 30.34°, 33.32°, 34.72°, and 39.01°. The physical mixture (PM) shows significant crystallinity with peaks at 8.59°, 10.72°, 14.48°, 17.48°, 19.11°, 20.35°, 23.49°, 25.28°, and 27.17°, accompanied by peak intensities of 551, 564, 1271, 2188, 1302, 2188, 1247, 1171, and 783, respectively. The kneaded mixture i.e. prepared cocrystals exhibits intense peaks at 8.61°, 10.76°, 10.99°, 11.35°, 13.46°, 14.17°, 14.48°, 17.50°, 17.95°, 19.09°, 20.37°, 23.47°, 25.28°, and 27.09°, with corresponding peak intensities of 579, 612, 562, 412, 531, 870, 1310, 2260, 1677, 1419, 2203, 1334, 1174, and 889. The appearance of new or shifted peaks at various 2θ angles, attributed to the components involved, provides evidence for the formation of a new crystalline phase, identified as a cocrystal ^{22, 23)} .

Fig. 3.

Overlay spectra of PXRD analysis of ITZ, proline, physical mixture (PM), and cocrystals.

Nuclear magnetic resonance (H ¹-NMR)

The H ¹-NMR spectrum of ITZ ( Figure 4) reveals aromatic hydrogen signals in the range of 6 to 8.5 ppm, as well as aliphatic alkyl halides between 6 and 7 ppm. Methylene groups appear at 0.5 to 1.8 ppm, and an aromatic CH is detected at 8.17 ppm. Additionally, signals corresponding to the dioxole heterocyclic ring with the triazole structure are present at 4.27 to 4.31 ppm. Proline’s spectrum shows the carboxylic acid group at 2 to 2.6 ppm, with a quadratic NH appearing between 3.2 and 4 ppm. The pyrrolidine ring produces signals between 1.5 and 4.9 ppm, specifically at 4.2 and 4.9 ppm. In the cocrystals, all key functional groups are observed along with some new peaks. Additional peaks between 3 and 4.7 ppm suggest the formation of NH-OH bonding, likely due to strong hydrogen bonding between the -NH group from ITZ and the -OH group from proline, which is further verified by FTIR analysis ²⁴⁾ .

In the present study, the ¹H-NMR spectra of the physical mixture and the cocrystal appeared nearly identical, with no significant changes in chemical shifts. This can be attributed to the fact that ¹H-NMR reflects the solution-state molecular environment, wherein both the physical mixture and cocrystal dissolve into the same constituent molecules of itraconazole and proline. Hence, distinct spectral differences are generally not expected. However, complementary solid-state characterization techniques such as FTIR, DSC, and PXRD provided clear evidence of cocrystal formation, demonstrating structural and physicochemical modifications that are not visible in solution-state NMR analysis.

Fig. 4.

H ¹-NMR spectra of ITZ, proline, physical mixture (PM), and cocrystals.

Raman spectroscopy analysis before and after stability studies

Raman spectroscopy can help determine whether a formed complex is a salt or a cocrystal. Proline has a favorable proton transfer capability due to its –NH⁺ and –COO⁻ functional groups. According to literature, pure proline exhibits a characteristic carbonyl stretching band at ~1,643 cm ⁻¹, which shifted to 1,615 cm ⁻¹ in our cocrystal spectrum. This shift of less than 30-40 cm ⁻¹ strongly suggests the formation of cocrystals ^{25, 26)} . In the present work, we did not separately acquire the spectrum of pure proline, but our observations are in good agreement with reported Raman data for proline. Importantly, neither the ITZ spectrum nor the physical mixture showed such a pronounced shift, further confirming the distinct molecular interactions in the cocrystal. Following stability studies, no additional peak shifts were observed in the Raman spectra, indicating the consistent stability of the prepared cocrystals, as shown in Figure 5.

Fig. 5.

Raman spectra of a) ITZ, physical mixture, and cocrystals before stability studies, b) ITZ, physical mixture, and cocrystals after stability studies.

Saturation solubility studies of the drug and prepared cocrystals

The saturation solubility of pure ITZ and its cocrystals was evaluated in simulated gastric fluid (SGF) at pH 1.2. The results demonstrated that the ITZ-proline cocrystal formulations significantly enhance solubility compared to pure ITZ. Specifically, pure ITZ exhibited a solubility of 8.75 µg/mL, while the physical mixture and cocrystals had solubilities of 24.43 µg/mL and 47.80 µg/mL, respectively. This indicates that the physical mixture achieved a threefold increase in solubility, likely due to hydrotropic effects, whereas the cocrystals produced through the kneading method showed a sixfold enhancement over pure ITZ. This increased solubility can be attributed to the zwitterionic form of proline, which is highly soluble in water. Furthermore, the cocrystals demonstrate a faster diffusion into the solution than pure ITZ, owing to their strong affinity for water molecules. As a result, the less soluble ITZ may become supersaturated in the solution, potentially forming an amorphous phase. This transition could contribute to an overall improvement in the kinetic solubility of the prepared cocrystals compared to pure ITZ ²⁷⁾ .

Determination of percentage yield and drug content of prepared cocrystals

Table 2 displays the percentage yield and drug content. The lower yield for the cocrystals may be attributed to material loss during the formulation process ²⁸⁾ . Visual inspections were also conducted to assess color, odor, appearance, and the ITZ content. Notably, no significant changes were observed in any of these parameters after the stability studies.

Table 2.

Percent yield and drug content during and after stability study of prepared cocrystal formulations (n= 3, Mean + S.D).

In vitro dissolution study

In vitro dissolution studies demonstrated that the ITZ-proline cocrystals exhibited a markedly improved dissolution rate compared to pure ITZ and the physical mixture. The cumulative drug release in simulated gastric fluid (SGF, pH 1.2) at 120 minutes was 11.36 ± 0.10% for pure ITZ, 25.08 ± 0.30% for the physical mixture, and 48.22 ± 0.20% for the cocrystals (n = 3). Statistical analysis (one-way ANOVA) confirmed that the dissolution of cocrystals was significantly higher (p < 0.05) compared to both pure ITZ and the physical mixture. These findings indicate that the enhanced dissolution rate of ITZ in the cocrystal formulation can be attributed to improved wettability and the hydrophilic, zwitterionic nature of the coformer ^{1, 29)} .

Although the physical mixture exhibited some dissolution improvement, the advantages of the cocrystals prepared by the kneading method were more pronounced. This was clearly observed in solid-state modifications confirmed through FTIR, DSC, and XRD analyses : in the physical mixture, most features of the individual components were retained, whereas the cocrystals displayed distinct band shifts, new endothermic events, and unique diffraction peaks, all indicating the formation of a new supramolecular structure. These structural changes result in better molecular interactions with the dissolution medium, leading to significantly higher drug release compared to the PM. Thus, while the PM provided only a limited benefit, the cocrystals offered a distinct thermodynamic and biopharmaceutical advantage.

Fig. 6.

In vitro dissolution studies of pure ITZ, physical mixture, and cocrystals in SGF (pH 1.2).

Stability study

Table 2 represents the drug content of the physical mixture and cocrystals, thereby reflecting the stability of the prepared cocrystals over the period of time.

In vitro antifungal activity

The antifungal activity of ITZ–proline cocrystals was evaluated against selected fungal strains and compared with pure ITZ and the physical mixture ( Figure 7). The cocrystals exhibited a significantly larger zone of inhibition at all tested concentrations (1, 3, and 5 µg/mL) compared to both pure ITZ and the physical mixture. For instance, at 5 µg/mL, the mean zone of inhibition was 28.3 ± 0.5 mm for cocrystals, 22.6 ± 0.4 mm for the physical mixture, and 20.1 ± 0.6 mm for pure ITZ (n = 3). Statistical analysis (one-way ANOVA) confirmed that the antifungal activity of cocrystals was significantly higher (p < 0.05) than that of the other groups.

This enhancement may be attributed to the improved solubility and dissolution rate of ITZ in the cocrystal formulation, which increases its bioavailability and antifungal efficacy ³⁰⁾ .

Our previous work (Shete et al., 2015) described the preparation and characterization of itraconazole cocrystals with several amino acids, including proline, using grinding methods, with preliminary evaluation of solid-state properties, dissolution, and antifungal activity. In the present study, we extend and refine those findings by focusing exclusively on the itraconazole-proline cocrystal and introducing several novel elements. A molecular docking-based virtual screening approach was used to rationalize the choice of proline as a coformer. Solid-state characterization was expanded to include ^1H-NMR and Raman spectroscopy, with the latter also confirming structural stability after three months of accelerated storage at 40°C/75% RH. Quantitatively, the proline cocrystal demonstrated a sixfold increase in solubility (47.8 vs. 8.75 µg/mL) and more than fourfold enhancement in dissolution (48.2% vs. 11.3% release in 120 min) compared to pure ITZ, which represents a substantial improvement over the threefold solubility enhancement reported earlier. Furthermore, antifungal activity was systematically reassessed at much lower drug concentrations (1-5 µg/mL), where the cocrystal exhibited significantly larger inhibition zones than pure ITZ, indicating potential for improved efficacy at reduced doses. Collectively, these additional studies provide deeper mechanistic insights and establish the ITZ-proline cocrystal as a promising strategy to overcome the solubility and bioavailability limitations of itraconazole.

Fig. 7.

Graphical presentation of a) zone of inhibition against Candida albicans at various concentrations, b) zone of inhibition against Aspergillus niger at various concentrations.

Conclusion

This study focused on the development of ITZ cocrystals with proline using the kneading method, specifically employing ethanol as a catalyst solvent. The kneading method is not only efficient and time-saving but also enhances product yield and crystallinity through controlled solid-state synthesis. Characterization in the solid state confirmed the successful formation of cocrystals, maintaining the compatibility and pharmacological properties of ITZ. Notably, the cocrystals exhibited a sixfold increase in solubility, enhanced dissolution rates, and greater antifungal activity against Candida albicans and Aspergillus niger compared to pure ITZ. Stability studies indicated that the cocrystals are stable, demonstrating stability for ITZ when cocrystallized with proline. Consequently, the strategy of preparing ITZ cocrystals offers a promising antifungal potential to combat fungal infections along with improved drug characteristics.

Acknowledgement

The authors are grateful to Krishna Vishwa Vidyapeeth (Deemed to be University), Karad, for providing the necessary funding and laboratory facilities to carry out this research work.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

Research data used to prepare this manuscript can be made available upon request to the corresponding author.

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