Praziquantel is an antiparasitic drug indicated for the treatment of the schistosomiasis disease. This drug has very low aqueous solubility, requiring high oral doses for its administration which gives rise to side effects, therapeutic noncompliance and the appearance of resistant forms of the parasite. Clay minerals, like sepiolite and montmorillonite, are innocuous, non-toxic, biocompatible and low-cost excipients. Additionally, clays have high adsorbent properties that allow them to encapsulate drugs in nanometric spaces present in the channels in the case of the sepiolite or between the layers in the case of the montmorillonite. The interactions between the drug and clay minerals are studied experimentally with the strategy for preparing interactions products in organic solvents (ethanol, acetonitrile and dichloromethane) so that the interaction will be more effective and will be enhanced the aqueous solubility of praziquantel. The results showed that in the interaction products, the drug interacted with both clay minerals, which produced the loss of the crystallinity of the drug demonstrated by different techniques. This led to a significant increase in the dissolution rate of the praziquantel in all the interaction products in the simulated gastrointestinal tract media, except for the praziquantel&#;montmorillonite product prepared in dichloromethane that presented a controlled release in acid medium. Moreover, in vitro cytotoxicity and cell cycle studies were performed in the interaction products prepared with ethanol. The interaction product with sepiolite was biocompatible with the HTC116 line cells, and it did not produce alterations in the cell cycle. However, interaction products with montmorillonite did not produce cell death, but they showed affectation and damage of cells in the cell cycle study at the highest concentration tested (20&#;100 µM). Therefore, the different organic solvents used are adequate for the improvement of the biopharmaceutical profile of praziquantel. Drug&#;clay interaction products, specifically with sepiolite, showed very promising results in which new accelerated oral release systems of the praziquantel were obtained.
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Hence, in this work, the preparation of interaction products is studied following the previously described procedure [ 33 , 34 ] comparing the use of different organic solvents, like ethanol, acetonitrile and dichloromethane. The aim is to prepare and characterize these systems and, by these means, to determine the potential influence of the organic solvent on the release and solubility of the drug. In particular, the results were compared with those obtained with ethanol in which a significant improvement in the biopharmaceutical profile was described. As well as this, in vitro cytotoxicity and cell cycle studies are carried out for checking if these systems are biocompatible and suitable for human use.
The interaction between PZQ and other excipients was also studied in order to improve the low solubility of the drug, for example, with calcium carbonate [ 18 , 19 , 20 ], β-cyclodextrins [ 21 , 22 , 23 ], polyvinyl-pyrrolidone [ 24 , 25 ], polyethylene glycols [ 26 , 27 , 28 ], sodium starch glycolate [ 29 ], phosphatidylcholine&#;containing liposomes [ 30 ], layered double hydroxides [ 31 ], among others. Specifically, the interaction between PZQ and montmorillonite in aqueous medium was previously studied, although no improvements of in vitro dissolution and absorption rates were observed [ 32 ]. The lack of relevant biopharmaceutical improvements was explained by the absence of PZQ in the interlayer space of montmorillonite. Water molecules present in the clay interlayer space would be blocking the entrance of the drug, thus PZQ would be solely absorbed in the external surface of montmorillonite. Therefore, in our previous studies [ 33 , 34 ], the interactions between PZQ and montmorillonite and sepiolite in absence of water were studied as a strategy for enhancing the aqueous solubility of the drug. To do this, ethanol 99% (v/v) was used as a solvent increasing the interaction between the drug and the excipient. The results showed a great increase in the dissolution rate of the drug and the total amount of drug dissolved in vitro in the interaction products, due to the high interaction between both components. Therefore, this new procedure was found to be an effective strategy to improve the biopharmaceutical profile of the PZQ.
Montmorillonite and sepiolite are innocuous, non-toxic, biocompatible and low-cost clay minerals. Additionally, clays have high adsorbent properties that allow them to encapsulate drugs in nanometric spaces present between the layers in the case of the montmorillonite or in the channels in the case of the sepiolite [ 12 , 13 , 14 , 15 , 16 , 17 ].
PZQ is the recommended treatment against all forms of schistosomiasis. It has high efficacy, low toxicity and is administered orally. In addition, the cost of production and sale of PZQ is low [ 2 , 6 ]. However, it has a few disadvantages. First, it is a racemic compound, with only one of its enantiomers being pharmacologically active [ 7 , 8 ]. In addition, it is a Class II drug of the Biopharmaceutical Classification System (BCS) and has very low aqueous solubility and high permeability, so that the dissolution is the factor limiting the rate of absorption [ 9 , 10 , 11 ]. Its low solubility in water makes it necessary to administer high oral doses, which gives rise to secondary effects that increase the therapeutic noncompliance and favor the appearance of resistant forms of the parasite. For these reasons, the use of low-cost natural inorganic excipients was proposed as a starting hypothesis to improve the aqueous solution profile of the PZQ, maintaining the final cost of the drug, which is very important given the population to which it is subject destined.
Schistosomiasis is the second neglected tropical disease with the highest prevalence, widespread mainly in tropical Africa. Therefore, the improvement of the existing treatment with the praziquantel (PZQ) drug is very important for saving lives. Since schistosomiasis is in the expansion phase and currently affects 240 million people in the world, of which only 88 million receive adequate treatment, it is estimated that it produces more than 200,000 deaths annually, and it is extended in 78 countries around the world, mainly in the tropical and subtropical region, being in Africa the second most prevalent disease in children. This disease is acquired by contact with fresh water infested with larvae of the parasite [ 2 , 3 , 4 , 5 ].
Neglected tropical diseases are infectious diseases that affect a sixth of the world population, more than a billion people, mainly in poor populations of tropical countries in Africa, Latin America, Asia and the Middle East. Their prevalence increases with the absence of salubrious water and adequate sanitation conditions and the most vulnerable population are children in contact with contaminated water [ 1 ].
HCT 116 cells were cultured in 24 well-plates (250,000 cells/well) in a final DMEM volume of 500 μL. In these conditions, cells were subjected to PZQ, VHS, SEP, IP PZQ&#;VHSet, and IP PZQ&#;SEPet samples for 48 h. Increasing concentrations of each sample (0.8 μM, 4 μM, 20 μM, and 100 μM) were used. Cells entering the apoptotic or necrotic cycle were detected with a propidium iodide staining procedure, as explained in a previous protocol [ 40 ]. Briefly, once the cells were in contact with each sample, they were collected and washed with 2 mL of phosphate-buffered saline (PBS) at 4 °C and subsequently fixed with ethanol 70° (V f = 1 mL, diluted 1:9 in PBS) for 5 min, while maintained on ice. Ethanol was withdrawn and fixed cells were washed with PBS. Then, they were resuspended in a solution consisting of 250 μL of PBS and 250 μL of a DNA extraction solution (0.2 M Na 2 HPO 4 , 0.1 M C 6 H 8 O 7 , pH 7.8) for 10 min at 37 °C. Then, the supernatant removed and 200 μL of the staining solution were added (8 μL propidium iodide (1 mg/mL) and 2 μL RNAse 100 (μg/mL)), the cells further incubated in the dark at 37 °C for 10 min. A FACScalibur cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA) was used to measure the fluorescence (FL2 detector). Sub-G1 cells (dead cells, that is, cells that entered either necrotic or apoptotic phases) were detected by the Cell Quest software (Version 1.0.2, BD, Biosciences, Franklin Lakes, NJ, USA).
AlamarBlue&#; bioassay (Thermo Fisher Scientific, Carlsbad, CA, USA) was used to study cell proliferation. To do so, HCT 116 cells were seeded in 96-well plates (density of 3&#;6 × 10 4 cells/cm 2 ) in a final DMEM volume of 200 μL. The corresponding solid samples were subsequently added (PZQ, VHS, SEP, IP PZQ&#;VHSet, or IP PZQ&#;SEPet). All samples were tested in a wide range of concentrations (100 μM, 20 μM, 4 μM, 800 nM, 160 nM, 32 nM, and 6.4 nM) and three replicates were used for each experience. Then, the well plates were incubated at 37 °C, 5% CO 2 for 48 h. At the end of contact time, 10 μL of cell viability reagent (PrestoBlue&#;, Thermo Fisher Scientific) was added to each well. The PrestoBlue&#; reagent is a resazurin compound that works as a cellular viability indicator since its color changes from blue to pink when reduced by living cells. After 15 min of incubation in presence of PrestoBlue&#; reagent, fluorescence was quantified at 535&#;90 nm in a microplate reader (Tecan, Zürich, Switzerland).
Cytotoxicity studies of PZQ, VHS and SEP were performed by using PZQ veterinary antiparasitic doses. Consequently, the amount of mineral (VHS and SEP) that should be subjected to study was five times higher than that of PZQ. To do so, intermediate dilutions of 100 mM and 10 mM were prepared in dimethyl sulfoxide (DMSO). The same procedure and concentrations were used for cytotoxicity studies of PZQ&#;VHSet and PZQ&#;SEPet.
Cellular viability and cell cycle profiles were studied over a human colorectal carcinoma cell line HCT 116 (ATCC ® CCL-247&#;, Manassas, VA, USA). HCT 116 cells were cultured in Dulbecco&#;s Modified Eagle&#;s Medium (DMEM, Gibco&#;, Dublin, Ireland) supplemented with 10% w/w of Fetal Bovine Serum (FBS, Gibco&#;, Dublin, Ireland), 1% of Glutamax (BioWhittaker ® , Cologne, Germany) and 1% of penicillin/streptomycin (BioWhittaker ® ). Cells were cultured in an incubator set at 37 °C and 5% of CO 2 .
The solubility of PZQ, PZQ&#;VHSet and PZQ&#;SEPet, which was used as a reference for the rest of the interaction products, were obtained in both HCl 0.001 M and SIF (phosphate buffer at pH = 6.8, without enzymes). PZQ solubility was obtained by placing 30 mg of drug in 10 mL of HCl and SIF (supersaturated conditions). The solution was stirred at 37 °C for 24 h (thermostatic bath). Then, the solution was centrifuged, the supernatant filtered (nitrocellulose membranes, 0.45 μm, Merck Millipore ® ) and quantified by HPLC (Agilent Techonologies Inc.). The amount of dissolved drug, determined by the chromatography, corresponds to PZQ solubility in the corresponding medium. In order to demonstrate the solubility improvement of PZQ in the composites (PZQ&#;VHSet and PZQ&#;SEPet), drug solubility was determined with the very same procedure. Triplicate experiments were performed in all samples.
Subsequently, release data fitting was performed using kinetic models intended to describe the drug release from immediate or modified dosage forms [ 35 , 36 , 37 ]. These models, such as zero order, first order, cube root, Higuchi, Peppas, and Weibull, were used to fit experimental data obtained from in vitro dissolution studies. The fitting was carried out linearizing the equations. The choice of the best model to describe the dissolution/release process of the drug was based on the correlation coefficient (R 2 ) criterium. A second evaluation criterium that is frequently used is called the Akaike Information Criterion (AIC) [ 38 , 39 ]. Herein, it is also considered.
During the in vitro dissolution tests, at predetermined time intervals, 5 mL of samples were withdrawn, the volume immediately replenished with fresh dissolution medium. The drug amount was quantified by High-Performance Liquid Chromatography (HPLC) after being filtered through 0.45 μm nitrocellulose membranes (Merck-Millipore ® , Darmstadt, Germany). HPLC was performed in a Infinity II HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a quaternary pump, autosampler, column oven and UV-VIS diode-array spectrophotometer. The stationary phase was a Lichrospher ® (100 RP-18, C18, 5 μm, 15 cm × 3.2 mm) column (Merck Millipore ® , Darmstadt, Germany). Isocratic conditions were used, the mobile phase formed by a mixture of H 2 O and CH 3 CN (35:65 v/v). The flow rate was set at 0.8 mL/min with a sample injection volume of 10 μL. PZQ was detected at 225 nm during the 5 min of the run time of the HPLC analysis. The software LC Open LAB HPLC (Agilent Technologies Inc.) was used to record and treat the chromatograms. The linear analytical method was obtained in the concentration range of 5 to 100 mg/L of PZQ in both dissolution media (correlation coefficients of 1 in both HCl 0.001 M and in SIF).
Hard gelatin capsules were subjected to the in vitro dissolution test. The capsules were prepared with 210 mg of each PZQ&#;VHS and PZQ&#;SEP interaction product, in which 35 mg corresponds to PZQ. Each of these interaction products (PZQ&#;VHS and PZQ&#;SEP) were prepared with the different solvents previously explained. Similarly, hard gelatin capsules containing 35 mg of PZQ were elaborated as reference.
In vitro dissolution tests were performed according to the European Pharmacopoeia procedures dealing with solid oral dosage forms. In particular, the paddle apparatus (apparatus 2, Sotax AT7, Teknokroma ® , Barcelona, Spain) with sinkers was set at 150 rpm and 37 °C, filled with 1 L of dissolution medium and working at sink conditions. Stomach physiological fluid was simulated by using a dissolution medium composed of HCl 0.001 M, while simulated intestinal fluid (SIF) was used with a phosphate buffer (pH = 6.8) without enzymes.
FTIR spectra were performed with the JASCO spectrophotometer (JASCO, Pfungstadt, Germany) which has an attenuated total reflectance (ATR) accessory. Measurements were performed in the range &#;600 cm &#;1 with a 0.5 cm &#;1 resolution and a well-plate sampler. The results were analyzed using the Spectra Manager II software (Version 2, JASCO).
The TGA was performed in air using a heating rate of 10 °C/min in the 30&#;400 °C temperature range and the DSC runs were performed with a heating rate of 2 °C/min and nitrogen was used as a purge gas in DSC under the flow of 15 mL/min.
Differential Scanning Calorimetric analysis (DSC) and Thermogravimetric Analysis (TGA) were performed with a Mettler Toledo mod. TGA/DSC1 calorimeter (Mettler Toledo, Barcelona, Spain). The TGA equipment sensor is a thermostatic microbalance with an accuracy of 0.1 μg, which is the limit established for weighing precision. On the other hand, the TGA/DSC1 has sensors that obtain the DSC signal directly and in real-time.
Powder X-ray diffraction was performed with equipment of Panalytical X&#;Pert Pro (Marvel Panalytical, Madrid, Spain) model, which is managed by a fully computerized system. It also has an automatic charger system and uses the X&#;Celerator detector (Marvel Panalytical) (linear type, detects the RX along a line) with the Cu Kα wavelength that allows a quick and accurate study. The information of diffraction was analyzed using X&#;Pert HighScore ® software (Version 2.2., Marvel Panalytical, Madrid, Spain).
The interaction products obtained were: PZQ&#;VHSet prepared with VHS and dispersed in ethanol, PZQ&#;VHSac prepared with VHS and dispersed in acetonitrile, PZQ&#;VHSdic prepared with VHS and dispersed in dichloromethane, PZQ&#;SEPet prepared with SEP and dispersed in ethanol, PZQ&#;SEPac prepared with SEP and dispersed in acetonitrile and PZQ&#;SEPdic prepared with SEP and dispersed in dichloromethane.
PZQ was put in contact with montmorillonite and sepiolite clay powders in 100 mL of different solvents: ethanol 99% (v/v), acetonitrile, and dichloromethane, in such way that the clay/drug ratio was 5:1 w/w. The dispersion was put into magnetic stirring for 24 h at room temperature. After 24 h, the solvent was evaporated at 40 °C using a rotary evaporator (Buchi ® R II, Flawil, Switzerland). The solid residue was stored in a desiccator at room temperature with freshly activated silica-gel grafted with a moisture indicator of Co salt.
Pristine racemic PZQ drug was purchased from Sigma Aldrich (St. Louis, MO, USA). Purified pharmaceutical degree. Montmorillonite (Veegum HS ® , VHS, Norwalk, CT, USA) was purchased from Vanderbilt Company (Norwalk, CT, USA). The Sepiolite samples from Vicálvaro (Madrid) (SEP) were kindly gifted by TOLSA (Madrid, Spain). Ethanol of 99% (v/v) of purity, acetonitrile, and dichloromethane were used as solvents.
The interaction products were prepared following the methodology described above in drug&#;clay. These materials and raw materials were characterized by powder XRD, showing the results in . PZQ drug revealed the most intense reflection at around 16.5° (2θ units) and other intense reflections at approximately 8.0°, 19.1° and 23.3° (2θ units) in accordance with that previously reported [41].
Open in a separate windowAfter interaction with the clay minerals, a practically complete absence of drug reflection was observed, probably due to a loss of crystallinity of PZQ ( ). In previous works, similar behavior of PZQ was observed in the formation of a solid dispersion with polyvinylpyrrolidone [24], with sodium starch glycolate [29], and with glycyrrhizic acid&#;forming micelles [42].
The interaction product of PZQ with sepiolite using different solvents in the preparation method ( a) showed an XRD pattern similar in all the cases. In the profiles, clear peaks of the PZQ drug were not observed; only the peaks of sepiolite were observed. Therefore, after the interaction, a loss of drug crystallinity was found.
In the oriented aggregate powder X&#;ray diffraction pattern of the interaction products of PZQ with montmorillonite ( b), the peaks of PZQ were not observed either in any of the products. However, the (001) reflection peak of the VHS increased in the interaction products, confirming the intercalation and the presence of PZQ in the interlayer space of the clay.
The interaction product PZQ&#;VHSet showed an increase in the interlayer space of montmorillonite from 1.26 nm to d(001) = 1.50&#;1.61 nm in accordance with that reported previously [33,43]. The interaction product PZQ&#;VHSdic increased to d(001) = 1.47 nm; and the spacing of PZQ&#;VHSac increased to d(001) = 1.51 nm ( b). These results corroborated that the PZQ is present in the interaction products and the peaks of PZQ were not observed probably due to the complete intercalation of the drug in the interlayer space of the clay and the loss of crystallinity of the drug. The PZQ molecules intercalate in the confined space of montmorillonite as individual molecules where no recrystallization is possible. Nevertheless, the d(001) reflection peaks are wider in the interaction products than in the pristine VHS. This indicates than a range of different d(001) spacings can be produced, especially in PZQ&#;VHSdic, where two peaks can be distinguished. In previous modeling studies, we observed the formation of a monolayer of PZQ in the interlayer space of VHS, where different densities of the drug can be found and several conformations of the adsorbate molecule can happen [44]. These differences can be responsible for the variation of d(001) spacing widening this (001) peak, maintaining the monolayer configuration.
The DSC profiles of the pristine PZQ showed a strong endothermic peak at 144 °C, according to previous work [18] ( ). The DSC profile of SEP and VHS indicated broad endothermic peaks in the range 50&#;90 °C due to the evaporation of water ( ).
Open in a separate windowIn a, the PZQ&#;SEPet and PZQ&#;SEPac interaction product profiles presented a very low-intensity peak corresponding to the melting of the drug. This indicates that a small amount of PZQ crystallized outside of the channels in the interstitial spaces of solid. Specifically, the enthalpy of the endothermic peak (ΔH) at 144 °C was 3.9 and 1.3 J g&#;1 respectively, which can be compared with the enthalpy of 90.4 J g&#;1 of the melting peak of the pristine drug. Therefore, the ΔH melting (PZQ&#;SEP)/ΔH melting (pure PZQ), was 4.3% in PZQ&#;SEPet and 1.4% in PZQ&#;SEPac. On the contrary, in the PZQ&#;SEPdic profile, the complete disappearance of the melting peak of the drug was observed.
The DSC curves of PZQ&#;VHS interaction products showed the water evaporation of the raw materials until 100 °C, and no melting peak of PZQ was observed in all interaction products (PZQ&#;VHSet, PZQ&#;VHSdic and PZQ&#;VHSac) ( b).
Therefore, the lack of the peak of PZQ in the interaction products was observed probably due to the amorphization and no recrystallization of the drug after the complete interaction with the clay. This justified the lack of drug reflections in the PZQ&#;clay powder XRD patterns. Previous studies found similar results [21,31,33].
In summary, the appearance of a small peak of the drug in some of the samples is practically insignificant and its appearance or non-appearance depends on the variability of production, compared with previous results [33]. Therefore, in drug&#;clay interaction products, a practically complete amorphization of the drug occurs.
According to the first weight loss of the TGA profile, absorbed water of VHS and SEP accounted for 8% and 10% w/w, respectively ( ). Zeolitic water loss of SEP occurred at 250&#;350 °C [45].
Open in a separate windowIn the interaction products both with sepiolite and montmorillonite, thermal decomposition of the drug resulted in weight losses in the range between 200 and 400 °C, corroborating the adsorption of the PZQ in these mineral solids ( ).
FTIR spectra of PZQ, SEP and VHS were previously described in detail [33,43,46,47]. In , the two most intense bands of the PZQ appear at &#; cm&#;1, which correspond to the ν(C=O) stretching vibration mode. The spectra of sepiolite and montmorillonite showed intense bands at &#;800 cm&#;1, corresponding to the ν(Si&#;O) and δ(MOHM&#;) (M, M&#; = Al, Mg) vibrations [47].
Open in a separate windowIn the interaction products prepared with sepiolite ( a) and montmorillonite ( b), the bands of the pristine PZQ were found, such as the bands that appeared at &#; cm&#;1, which are characteristic of the δ(CH) bands of bending vibrations. This corroborated that the drug is present in the drug&#;clay interaction products regardless of the solvent used in the elaboration process, but the bands of the PZQ are masked by the clay in all cases, since the clay is in a much higher proportion.
Drug release profiles of the interaction products with sepiolite (PZQ&#;SEPet, PZQ&#;SEPac and PZQ&#;SEPdic) and montmorillonite (PZQ&#;VHSet, PZQ&#;VHSac and PZQ&#;VHSdic) compared with the pristine PZQ were studied in sink conditions in acid aqueous medium with HCl 0.001 M (pH = 3) and in simulated intestinal fluid (pH = 6.8) ( ).
Open in a separate windowThe PZQ&#;SEP interaction products in acid medium revealed that all the products increased the dissolution rate of the PZQ drug. This enhance in the drug release profile is higher in the following order PZQ&#;SEPdic > PZQ&#;SEPet > PZQ&#;SEPac ( a). The same results were obtained in the SIF medium ( b). Therefore, the interaction products with sepiolite increase the dissolution rate of the PZQ greatly using any of the three solvents in the preparation method, with PZQ&#;SEPdic being the one that showed the best results in both media. This higher dissolution rate is due to the loss of crystallinity of PZQ that occurred during the intercalation in sepiolite. The amorphization overcomes the high cohesion energy in the packing of the PZQ crystal lattice [43] needed for its dissolution. In particular, this amorphization is higher in PZQ&#;SEPdic ( a) and, hence, its dissolution rate is the highest one.
The PZQ&#;VHS interaction products showed differences between them in acid medium ( c). In this medium, a strong increase in the dissolution rate of the drug was observed in the PZQ&#;VHSac product, whereas PZQ&#;VHSet showed only a slightly higher dissolution rate than the pristine PZQ. On the contrary, a surprising result of PZQ&#;VHSdic was found in acidic medium. In this case, the drug is released at a constant rate. This, in principle, leads to better control of plasma concentration and offers several advantages. Therefore, the PZQ&#;VHSdic in acidic medium showed a release that is interesting for new PZQ controlled release systems ( c). In simulated intestinal fluid, all the PZQ&#;VHS interaction products showed an increase in the dissolution rate with respect to the pristine PZQ drug. The material prepared with acetonitrile (PZQ&#;VHSac) presented the fastest release profile, followed by PZQ&#;VHSet and PZQ&#;VHSdic, which also presented a similar profile. In general, the increase in the dissolution rate of the PZQ&#;VHS interaction products also owes to the amorphization of PZQ. The peculiar behavior of PZQ&#;VHSdic at low pH can be due to a possible effect of the pH on the opening of the pores and interlayers of VHS that does not occur at higher pH, where the swelling is faster. Among the solvents used in this work, the dichloromethane is the only solvent non-miscible with water. It is likely that this behavior changed the macroscopic structure of the solid.
Moreover, in general, the PZQ&#;clay dissolution rate was higher in acidic medium (pH = 3) than in simulated intestinal fluid (pH = 6.8), in concordance with the results in no-sink conditions previously obtained in PZQ&#;clay interaction products prepared with ethanol [33]. Therefore, the interaction of the drug with the clay minerals induced an increase in dissolution rates with the independence of pH. This improvement was demonstrated in all interaction products with sepiolite in both media, and with montmorillonite in SIF medium. The interaction products with montmorillonite in acid medium showed a different behavior between them, with the PZQ&#;VHSac being the one with the highest dissolution rate.
Therefore, according to the results obtained in vitro, PZQ&#;VHS and PZQ&#;SEP interaction products might improve the oral bioavailability of the drug by increasing both dissolution rate and amount of drug dissolved in the medium that simulates the stomach, obtaining new accelerated oral release systems of the PZQ. Moreover, the PZQ&#;VHSdic in the acidic medium could be a new controlled release system of the drug.
Subsequently, the experimental dissolution data of PZQ, PZQ&#;SEP and PZQ&#;VHS interaction products were fitted to various kinetic models in order to analyze the drug release. The correlation coefficient (R2) and Akaike Information Criterion (AIC) obtained from the dissolution model&#;s fitting are summarized in Tables S1 and S2. In general, the results showed that zero-order and first-order models were not appropriate to study these dissolution kinetics. In the same way, Square Root (Higuchi) and Power Law (Peppas) are not appropriate for the adjustments of the experimental dissolution values since there are no sufficient values lower than 63.2% of the drug release to adjust a kinetic [37]. In Tables S1 and S2, correlation coefficient and AIC values of PZQ, PZQ&#;SEPac, PZQ&#;SEPdic, PZQ&#;SEPet, PZQ&#;VHSac, PZQ&#;VHSet in acid and SIF media suggested that the Weibull model could be considered as an adequate model to describe the release kinetic, because the R2 obtained was the highest value and AIC value was the lowest once compared with the rest of the proposed models. The Weibull model presented an initial burst release [48,49], which is increased in the interaction products with clays. However, PZQ&#;VHSdic in an acid medium (pH = 3) showed a different dissolution profile, as can be seen in Table S2 and c. In this case, the results suggested that the Cube Root (Hixson&#;Crowell) is the more accurate model to describe the release kinetics. Therefore, the PZQ&#;VHSdic IP in an acid medium (pH = 3) presented a release that is controlled by the dissolution rate of the drug particles and it is assumed that the particles of the interaction product are isometric and monodisperse. This geometric shape of the particles remains constant and there is a decrease in the surface area associated with the dissolution of the pharmaceutical form [50,51].
The PZQ&#;SEPet and PZQ&#;VHSet showed an increase in the solubility compared to the pristine drug in the studied media. The results in the acid aqueous medium with HCl 0.001 M (pH = 3) indicated that the solubility of the PZQ&#;SEPet and PZQ&#;VHSet interaction products were similar in both cases (0.71 and 0.74 mg/mL, respectively). Similar results are obtained in simulated intestinal fluid (pH = 6.8), where the solubility of PZQ&#;SEPet and PZQ&#;VHSet was 0.61 and 0.65 mg/mL, respectively. Therefore, the interaction products enhanced the solubility of the drug, and this increase was in the range of 36&#;48% with respect to pristine PZQ ( ).
In vitro cytotoxicity tests in the HCT116 cell line were performed in PZQ, SEP, VHS, PZQ&#;SEPet and PZQ&#;VHSet products. The interaction products with ethanol were selected as a reference from the rest because they present an increase in the in vitro dissolution profile in sink conditions and in non-sink conditions, as demonstrated by previous work [33].
The raw material (PZQ, SEP and VHS) in concentrations between 6.4 nM and 100 μM were tested, demonstrating that they can be considered biocompatible toward HCT116 cell lines due to the fact that the percentage of the cell viability was slightly lower than the untreated cells in all cases (control) ( ).
Open in a separate windowIn the PZQ&#;SEPet interaction products, the loss of cell viability was not observed in any of the concentrations tested, even a positive effect in cell growth was observed with respect to the control, and pure SEP and PZQ. Therefore, the PZQ&#;SEPet revealed its biocompatibility with cell and had a positive effect on the cell viability compared to raw PZQ and SEP solids in these ranges of concentrations ( a). PZQ&#;SEPdic and PZQ&#;SEPac products are expected to behave similarly.
PZQ&#;VHSet showed a slight loss of cell viability like pure products, up to a maximum of 11% in the highest concentration tested. In this case, a positive effect on cell growth was not observed, but the viability of the PZQ&#;VHSet interaction product was similar to that of the pristine drug and VHS, therefore it can be considered biocompatible according to the in vitro results ( b). Likewise, PZQ&#;VHSdic and PZQ&#;VHSac products are expected to have similar behavior.
The cell cycle tests by means of propidium iodide were performed to investigate the cell cycle corresponding to the Sub-G1 phase of the cells in contact with the interaction products prepared (PZQ&#;SEPet and PZQ&#;VHSet) and their raw materials (PZQ, SEP and VHS) ( ). These studies allowed us to deduce if there is any alteration of any phase of the cell cycle despite the cell proliferation not being affected, after observing above that there is no significant loss in cell viability of the cells treated with the samples studied.
Open in a separate windowThe control tests are shown in the upper part of the figure ( ). Control tests demonstrated that, in comparison with cellular cycles of healthy, untreated cells (&#;not treated&#; cells in ), DMSO does not affect the cells, unlike etoposide (antineoplasic drug). As can be seen by the cell cycle analysis, the etoposide control group is severely affected and induces death to 64.3% of the cell population.
In a, the results showed that the cell cycle was not affected when the cells were treated with PZQ, SEP and PZQ&#;SEPet, and only at high concentrations, the cell death was increased slightly, although not in a notable way. Furthermore, in none of the concentrations tested was it observed that the PZQ&#;SEPet interaction product produced a significantly higher percentage of cell death than that with the pure PZQ.
The results of PZQ, VHS and PZQ&#;VHSet samples are shown in b. The PZQ&#;VHSet interaction product showed that the cellular cycle of the cells was not affected, and cell death was not observed at low sample concentration (0.8&#;4 µM). At high concentrations (20&#;100 µM), affectation of the cell cycle of the cells was observed as well as greater cell death. This damage in the cell cycle is very high at the 100 µM concentration. This is the same in pure VHS clay, therefore, this occurs in the interaction product due to the presence of the clay mineral.
In summary, the PZQ&#;SEPet interaction product showed that it does not affect, in any concentration, the cellular cycle of the cells, and its behavior is similar to the pure drug. However, the PZQ&#;VHSet interaction product showed that it affects, at high concentrations, the cellular cycle, so only the lower concentrations (<0.8&#;4 µM) do not affect the Sub-G1 phase of the cells ( ).
In a subsequent work, the same PZQ-HH was also found by fast cooling crystallization of PZQ in the presence of MeOH/HO mixtures with 30 and 40%of HO and in EtOH/HO mixtures with 40 and 50%of HO [ 62 ].
In addition, IR analysis showed a band at cm, not visible in the case of anhydrous Form A and like the peak observed by Zanolla and coworkers ( cm), which was attributed to the C-OH signal typical of hydrates [ 64 ]. Moreover, some shifts of the C=O bands at &#; cmwere seen, like polymorphs B and C of PZQ, which were related to a change in the conformation of PZQ molecules fromto
The PXRD pattern of the new solid was indexed using PDXL2 software, and the solution with the best reliability was a monoclinic space group of P2/ m with a unit cell volume of .09 Å 3 (a = 14.128 Å, b = 13.536 Å, c = 13.187 Å; α = γ = 90°, β = 112.368°) and a Z value of five for the unit cell. However, the scCO 2 process did not generate single crystals for SXRD analysis; therefore, no crystal structure was solved and deposited in the CSD.
The novel form showed a complex thermal profile by DSC: the first endotherm with onset at 83.11 °C (peak 92.5 °C) was related to dehydration into a novel dehydrate (confirmed through variable-temperature PXRD); then, the novel dehydrate melted and recrystallized to Form A; the final endotherm occurred at 139 °C, agreeing with the melting of Form A. The TGA experiment detected a mass loss of 3.38% w / w in correspondence with the first endothermic event of DSC; also, KF titration attested to a water content of 3.24% w / w , in close agreement with the mass observed in TGA, confirming that the isolated solid was a hydrate with 0.6 equivalent (eq.) of water and, more precisely, a hemihydrate with 0.1 eq. excess water due to the hygroscopicity of the material.
A later article reports another hydrated form found through supercritical COprocessing. Specifically, MacEachern and coworkers observed the formation of the new phase using rapid expansion of supercritical solution (RESS) as a supercritical carbon dioxide (scCO) processing method [ 65 ]. PZQ was wrapped in a Kimwipe and carefully placed in a 100 mL high-pressure vessel in an extractor oven (±0.5 °C). The vessel was pressurized with COusing an SFT-10 pump at 24 mL/min and sealed by closing the valves at the inlet and outlet. The vessel was held at 40 °C/17.9 MPa for 22.5 h and, after this period, purged with COat the set pressure for 20 min at approximately 4&#;5 L/min. The pump was then stopped, and the vessel was de-pressurized. The solubilized solids were collected in the collection vials through a rapid expansion from the supercritical solution (RESS) process. The solid remaining in the Kimwipe after the experiment was simply exposed to high-pressure conditions for the duration of the experiment.
In the same work, the authors also evaluated the effect of the thermal stress on PZQ-MH and PZQ-HH dissolution behavior. For this aim, the hemihydrate was heated at 84 and 124 °C for a period of 20 min (hereinafter referred as HH84 and HH124), whereas the monohydrate was kept at 124 °C for 20 min (referred as MH124). DSC and PXRD analyses of the three samples confirmed what was already clear from HS-ATR-MIR coupled to MCR-ALS: HH84 evidenced the presence of Form B with a small amount of amorphous (visible from the small baseline shift in PXRD); HH124 and MH124, instead, revealed the predominant presence of Form A, without traces of Form B and a little amount of amorphous. Considering the influence of this thermal stress on the dissolution of the solids, HH84 showed both an increase in the amount of drug dissolved at early times (10 min) and an increase in the dissolution rate in comparison to PZQ-HH. On the contrary, HH124 and MH124 presented similar behavior: early dissolution times due to the presence of the amorphous part, but a lower overall dissolution driven by the presence of Form A.
As for PZQ-MH, it is known that its dehydration undergoes a single-step transformation, directly converting into Form A [ 63 ]. Under the same above-mentioned HS-ATR thermal treatment, the MH dehydration process started approximately after 70 °C, which resulted in the concomitant formation of Form A and reached a maximum around 90 °C (90%). Then, the melting event of Form A occurred and became relevant after 125 °C. All the events detected were in full agreement with DSC observations. As in the case of PZQ-HH, the first change in the MIR spectra was the disappearance of the signal assigned to ν(OH) at cm, due to water loss during the conversion of PZQ-MH into Form A. Simultaneously, the ν(C=O) of the amide group ( cm) split in two vibrations with displacements to higher ( cm) and lower ( cm) wavenumbers, in accordance with the signals detected for Form A.
For PZQ-HH, it was previously established through DSC analysis that the dehydration initially generates polymorph B and, consequently, Form A appears [ 64 ]. Under HS-ATR thermal treatment, indeed, PZQ-HH totally converted into Form B at 90 °C; then, a second transformation occurred at 107 °C which quantitatively yields Form A at about 115 °C. Further heating gave rise to the last transition, observed at 136 °C, which corresponds to the complete melting of Form A. The MIR spectra, simultaneously acquired during the ATR stage, revealed a first change in the disappearance of the signal at cmdue to the loss of water during the transformation of PZQ-HH to Form B. Simultaneously, the splitting of ν(C=O) of the amide group ( cm) into two different vibrations ( and cm) and the displacement of one of them to a higher wavenumber were observed, showing the gain of rigidity in the covalent bonds and confirming the transformation into Form B. Subsequently, the conversion from Form B to Form A was displayed by the increasing of ν(C=O) of the amide groups, shifting from and cmto and cm, respectively.
Considering these two above-discussed new hydrated forms, both PZQ-MH and PZQ-HH solid-state transformations under heat stress were monitored through hot-stage attenuated total reflectance coupled to mid infrared spectroscopy (HS-ATR-MIR) empowered by multivariate curve resolution coupled to alternating least squares (MCR-ALS) and DSC in a study reported by Salazar-Rojas et al. [ 103 ]. The monitoring setup involved heating the samples on the ATR stage at a rate of 5 °C/min (30&#;145 °C), while acquiring MIR spectra (&#;600 cm) every minute. MCR-ALS was used to resolve the high spectral overlapping evidenced among the species.
Interestingly, other authors noticed that PZQ-HH could also be obtained starting from PZQ monosolvate with acetic acid (PZQ-AA) by putting the latter in a water vapor atmosphere: in this condition, desolvation of AA and solvation of HO occurred [ 62 ].
Considering biopharmaceutical properties, PZQ-HH demonstrated improved water solubility (310.89 ± 3.07 mg/L) compared to Form A (217 ± 10.33 mg/L). Moreover, the IDR was of 0. ± 0. mg cm, like that attested for Form B (see Section 4 ) and twice that of the commercially available Form A (0. ± 0.031 mg cm). As for the in vitro activity against adult, PZQ-HH exhibited an ICof 0.15 μM, identical to Form A (ICof 0.1 μM) [ 97 ].
The FT-IR spectrum of PZQ-HH presented a very sharp band at cm &#;1 due to the presence of H 2 O, differently from the anhydrous Form A. In the C=O stretching region, a single shifted broad band was detected at cm &#;1 , instead of the typical doublet at and cm &#;1 of Form A, confirming the intermolecular interactions between the drug and water via PZQ carbonyl groups.
The 13 C CPMAS SSNMR spectrum confirmed the formation of a new pure phase and the presence of only one molecule in the ASU, as attested by crystallographic data. SSNMR assignments for Cq indicated high similarities between PZQ-HH and PZQ Form B, while having significant differences from Form A.
The PXRD pattern of PZQ-HH was completely different from the already known PZQ solid forms: it presented characteristic sharp reflections that did not overlap either with those of Form A or with Form B and C and the enantiomeric hemihydrates. From the capillary PXRD pattern, the crystal structure of the new solid (indexed as WUHQAU in the CSD) was solved: PZQ-HH crystallizes in the triclinic space group of P-1 (as Form A), but just one independent molecule, rather than four as for TELCEU, is present in the ASU ( Figure 6 ). In the crystal unit cell, every HO molecule is linked to two PZQ entities (i.e., one (R)- and one (S)-) through H-bonds, confirming the 1:0.5 stoichiometry, and PZQ carbonyls exhibit the most common-conformation (similarly to B and C polymorphs and differently from Form Aconformation).
The DSC curve of the novel solid presented a sharp dehydration endotherm at about 68 °C and two other endothermic events at 109.05 °C and 133.95 °C, attributable to the melting points of polymorph B and Form A, respectively. The same events were confirmed by hot-stage microscopy (HSM). Additionally, TGA was performed to evaluate the sample weight loss that was of 2.19%, corresponding to the theoretical value for a hemihydrate.
The formation of PZQ-HH was also promoted by grinding PZQ Form A in the presence of seeds of preformed PZQ-HH (95:5 or 90:10 w / w ) for 1 h of LAG with H 2 O: a few seeds of the hydrate were sufficient to enhance the complete transformation of Form A in PZQ-HH, giving the chance to Form A to convert into PZQ-HH in a one-step procedure, instead of the above-mentioned two-step mechanochemical treatment.
In the same year of appearance of the above-described PZQ-MH, Zanolla and coworkers reported a novel PZQ racemic hemihydrate (PZQ-HH) obtained through mechanochemistry [ 64 ]. Several methods to achieve this hydrated form are reported in their work: (i) PZQ-HH could be produced starting from the commercial Form A through a two-step mechanochemical treatment (i.e., 30 min of NG and, subsequently, 1 h of LAG with HO), passing through the formation of an amorphous intermediate; (ii) it could also be obtained in a one-step 1 h LAG process when using Form B as starting material; (iii) its formation was also observed via slurry experiments of PZQ polymorph B in three days (contrary to Form A, which never converted to PZQ-HH with the same technique).
Starting from the thermal analyses, the DSC curve of the new form exhibited two endothermic transitions: the first, as a large endothermic event&#;without any defined peak&#;starting at about 78 °C and ending at 130 °C, was attributed to the dehydration, and the second (144 °C) was assigned to the PZQ Form A melting point. TGA experiments showed a gradual weight loss in the large range of 80&#;120 °C (total of 5.4%, agreeing with the theoretical value for a PZQ monohydrate). Additionally, the water content of PZQ-MH was evaluated through the official USP loss on drying test [ 102 ], at 105 °C up to constant weight (3 h), where the sample weight loss was of 94.6%, confirming the 1:1 stoichiometry. Sharp peaks clearly different from those of Form A were detected through PXRD, confirming the crystallinity of the sample. Passing to spectroscopic properties, PZQ-MH showed an intense peak at cm, confirming the presence of water in the crystal structure; in the region related to the carbonyl stretching vibration, the monohydrated form exhibited a single shifted carbonyl signal at cm, compared to the previously mentioned double peak at and cmof anhydrous Form A, assuming that the carbonyl group is involved in the interaction with water, also visible from the deshielding of the carbonyl carbon atoms in SSNMR spectra.H NMR spectroscopy testified to the presence of water with an easily distinguished resonance at δ 1.63 ppm: the integral of the signal was compared to the doublets resonating in the 4.6&#;3.7 ppm region and used to check the 1:1 stoichiometry.
First, PZQ hydrate was reported by Salazar-Rojas and coauthors, who obtained a monohydrate (PZQ-MH) as a peculiar pink solid: PZQ Form A was melted in a vacuum oven at 150 °C with 60 mmHg for 30 min; then, it was transferred to a humidity temperature-controlled chamber while being whisked and rapidly cooled with liquid nitrogen at 20 °C and 70% of relative humidity (RH %) [ 63 ]. The obtained solid was kept in a freezer (&#;20 °C) overnight and then stored below 30 °C in a closed flask for subsequent characterization.
The study of hydrated solid forms is of great importance as water is often present during different preformulation studies and several stages of drug manufacturing. Water molecules contain both donor and acceptor hydrogen groups and show, therefore, the capability to form H-bonds with other compounds. One of the direct implications of hydrate formation is the modification of several physicochemical properties of the drug. Indeed, hydrated forms are known to be less soluble than the corresponding anhydrous drug, thus having an impact on its biopharmaceutical properties [ 101 ]. Based on that, a comprehensive study on the hydrated forms of a particular drug is often necessary.
Later, Guedes Fernandes de Moraes et al. reported the discovery of a dimethylacetamide (DMA) solvate of PZQ [ 62 ]. Specifically, PZQ-DMA solvate was obtained through fast cooling crystallization of PZQ Form A: two different levels for the initial concentration of PZQ (low and high) and two for the cooling rate [slow (0.2 °C/min) and fast (5 °C/min)] were investigated and PZQ-DMA solvate was obtained with low and high concentrations of PZQ but just with rapid cooling crystallization, whereas the slow cooling method gave the same starting Form A. The authors observed a completely different PXRD pattern from that of TELCEU or other reported PZQ solid forms, and thermal analyses were essential to prove that the new form was a solvate. The DSC curve showed an endothermic event attributable to desolvation at 58 °C, followed by two melting events corresponding to the melting of Form G and the melting of Form A, respectively (see Section 2.1 and Section 4 ). The desolvation was also confirmed through the TGA experiment by a weight loss of approximately 20%, agreeing with the theoretical value for a monosolvate. Moreover, SEM images demonstrated the presence of lath-shaped crystals for the new solvate, differing from the smaller acicular crystals of Form A ( Figure 4 ).
Passing to the 13 C CPMAS SSNMR spectra of PZQ-2P and PZQ-AA, the presence of the solvent molecules was easily detected by peaks at 172.1 (COOH) and 19.7 (CH 3 ) ppm for PZQ-AA and at 177.5 (C=O) and 19.4, 28.9, and 40.4 (CH 2 ) ppm for PZQ-2P. Additionally, in the case of PZQ-AA, one of the carbonyl signals was not visible due to the overlapping with the COOH signal of AA (172.1 ppm), whereas it was recognizable in PZQ-2P. In both spectra, the number of signals was consistent with the 1:1 stoichiometry.
Considering FT-IR results, PZQ-2P and PZQ-AA displayed the stretching of the heterocyclic carbonyl (originally at cm &#;1 in Form A) at cm &#;1 , thus showing a lower frequency difference in the carbonyl vibrations than that observed for Form A and confirming the presence of an anti -conformation. Moreover, the ν(N-H) band, originally present in 2-pyr at &#; cm &#;1 and not present in pristine PZQ, resulted in a downward shift in the solvate ( cm &#;1 ), confirming both the presence of 2-pyr in the structure and the intermolecular interaction between the two coformers. As for PZQ-AA, two bands detected at cm &#;1 were helpful in confirming the presence of AA in the structure of the solvate.
PXRD patterns of the two solvates showed reflections clearly different from PZQ Form A and other known PZQ solid forms, being, instead, quite similar and suggesting isostructurality between the two new forms. Synchrotron X-ray diffraction, through which their crystal structures were solved, confirmed that the solvates are isostructural: they both crystallize in the triclinic space group of P-1, showing one PZQ molecule linked via a strong H-bond to one molecule of solvent in the ASU ( Figure 7 ). Intermolecular interactions and stoichiometry were confirmed through FT-IR and SSNMR analyses.
The first two PZQ solvates were discovered through a mechanochemical screening performed with several liquids [ 66 ]. Specifically, 2-pyrrolidone (2-pyr) and acetic acid (AA) were found to be suitable liquids to give two PZQ solvates (PZQ-2P and PZQ-AA) with a 1:1 stoichiometry by grinding the drug in the presence of each solvent, regardless of the quantity added (η values tested in the range 0.05&#;0.5 μL [ 105 ]), for 30 min at 25 Hz. The same outcome was observed when starting from anhydrous polymorph B of PZQ.
It is well known from the literature that solvates are usually discovered either by chance during a specific manufacturing process or via systematic polymorph screening programs such as solution crystallization, slurry, and, more recently, mechanochemistry [ 104 ].
Cocrystallization represents a growing strategy to obtain new crystal forms in the context of pharmaceutical science [ 106 ]. Indeed, pharmaceutical cocrystals, formed by an active pharmaceutical ingredient (API) and one or more cocrystal formers, are highly interesting due to their effect on physicochemical properties (i.e., solubility, bioavailability, mechanical/humidity/thermal stability, and compressibility) and their role in separation technologies, particularly for chiral molecules [ 107 115 ].
Considering that the molecular structure of PZQ does not contain salt-forming functional groups (but just two carbonyl groups acting as H-bond acceptors) and the possibility to create diastereomeric cocrystal pairs for its chiral resolution, PZQ&#;s propensity for cocrystal formation has recently attracted great attention.
&#;1 and were shifted to larger wavenumbers in the cocrystals, requiring higher energy for the stretching, which is consistent with the formation of weaker interactions (i.e., C-H···O and O-H···O).Espinosa-Lara and coworkers explored for the first time PZQ&#;s cocrystallization tendency by combining (RS)-PZQ and aliphatic dicarboxylic acids via LAG, assuming that cocrystallization might be induced by the formation of dimeric heterosynthons [ 58 ]. Specifically, PZQ was combined with oxalic acid (OXA), malonic acid (MALO), succinic acid (SUC), maleic acid (MALE), fumaric acid (FUM), glutaric acid (GLU), adipic acid (ADI), pimelic acid (PIM), suberic acid (SUB), azelaic acid (AZE), and sebacic acid (SEB) in a 2:1, 1:1, and 1:2 molar ratios in two different solvents (i.e., AcT and ACN). Only SUB, AZE, and SEB did not allow PZQ cocrystallization and nine new cocrystals were discovered. Comparison of the PXRD patterns indicated that OXA, MALO, SUC, MALE, FUM, and GLU generated a 1:1 cocrystal (i.e., PZQ-OXA, PZQ-MALO, α-PZQ-SUC, PZQ-MALE, PZQ-FUM, and PZQ-GLU), while ADI and PIM gave a 2:1 cocrystal (PZQ-ADI and PZQ-PIM). Also, all FT-IR spectra did not correspond to a sum of the starting materials and significant shifts were seen in PZQ carbonyl vibration bands, reflecting the various H-bonding patterns of the cocrystals. The C=O stretching vibrations of the starting coformers ranged from to cmand were shifted to larger wavenumbers in the cocrystals, requiring higher energy for the stretching, which is consistent with the formation of weaker interactions (i.e., C-H···O and O-H···O).
To solve the crystal structure of the new solids, PZQ and each coformer were also dissolved in 2:1, 1:1, 1:2, 1:3, and 1:4 molar ratios in hot AcT or ACN in the presence of a small quantity of mechanochemically preformed cocrystals as seeds to perform classical solution crystallization. This procedure allowed the isolation of single crystals suitable for SXRD analysis in the case of PZQ-OXA, PZQ-MALO, PZQ-MALE, PZQ-FUM, PZQ-GLU, and PZQ-ADI. PZQ-PIM, instead, did not give single crystals and, therefore, its crystal structure was not solved. In case of using PZQ and SUC as coformers, solution crystallization provided single crystals showing different PXRD patterns from that observed with α-PZQ-SUC, thus giving rise to a polymorphic form, namely β-PZQ-SUC ( Figure 8 ), as also attested to from the similarity of the two FT-IR spectra.
PZQ-OXA, β-PZQ-SUC, and PZQ-GLU (indexed in the CSD as TELCOE, TELDAR, and TELDIZ, respectively) crystallized in the same space group (P-1) and had similar unit cell edge lengths, suggesting similarities in the supramolecular organization of the three cocrystals: each of the COOH group of the acids was connected to a PZQ molecule through double or triple bridged heterodimeric motifs containing O-H···O H-bonds and C-H···O contacts. The ASU of the three cocrystals comprises one molecule of PZQ with carbonyls in the
anti
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-conformation and one molecule of each coformer, confirming the 1:1 stoichiometry already established by PXRD. In PZQ-SUC and PZQ-GLU, PZQ molecules were bound together through double-bridged homodimeric motifs.PZQ-FUM (refcode TELBUJ in the CSD) crystallizes in the space group of P-1 and shows analogous connectivity of PZQ-OXA. An important difference was that, in the case of PZQ-FUM, there were two sets of independent molecules in the ASU.
PZQ-MALE (TELCIY in the CSD), instead, crystallizes in the orthorhombic space group Pna21 and shows a connectivity like PZQ-SUC, with additional C-H···O contacts between MALE molecules. Both PZQ-FUM and PZQ-MALE displayed an
anti
-conformation of carbonyls of PZQ molecules.Somewhat different was the case of PZQ-MALO (indexed as TELDEV in the CSD): this cocrystal crystallized in the P21/
c
space group and was the unique new system which presented the samesyn
molecular conformation of PZQ Form A. This configuration allowed for the formation of 26-membered cyclic [2 + 2] aggregates: the variability in the connectivity occurred because MALO entities were disordered over two positions, giving rise to [2 + 2] assemblies with slightly different conformations. Additionally, MALE molecules were bound together to further stabilize the crystal structure.PZQ-ADI (refcode TELCAQ in the CSD) crystallizes in the P-1 space group with a 2:1 stoichiometry; therefore, the larger quantity of PZQ favored the formation of homodimeric motifs (PZQ-PZQ entities) to form double chains linked to the coformer. In case of PZQ-ADI, a low temperature crystal structure was also indexed (refcode TELCAQ01), which confirmed that the crystal structure was quite compact.
To summarize, in all cocrystalline structures, the dominant H-bonding interactions consisted of heterodimeric motifs formed between PZQ and dicarboxylic acids. Seven of the nine motifs were characterized by O-H···O H-bonds and occurred with either of the C=O groups of PZQ. Additionally, PZQ molecules were bound together through homodimeric H-bonds and there was only one case showing double-bridged H-bonding contacts between molecules of the cocrystal former, that is, PZQ-MALO. Worthy of notice is that the
anti
-conformation was adopted for all new cocrystals, except for PZQ-MALO, which exhibited the samesyn
conformation of (RS)-PZQ Form A.Physicochemical properties (i.e., thermal and spectroscopic behavior, solubility, dissolution), oral bioavailability, and pharmacokinetic parameters of some previously discussed cocrystals were deeply investigated by Wasim et al. [ 116 ]. Precisely, their aim was to elucidate the relationship between the physicochemical properties and pharmacokinetic parameters (i.e., Cmax, Tmax, and area under the curve, AUC) of PZQ cocrystals and the chain lengths (=n) of the selected dicarboxylic acid coformers, namely OXA (n = 0), MALO (n = 1), SUC (n = 2), GLU (n = 3), and ADI (n = 4). In the same work, they also assessed the effect of a polymer (hydroxy propyl cellulose, HPC) on PZQ cocrystal formation.
Firstly, cocrystals were prepared using the slurry crystallization method, and the results were evaluated by comparing their PXRD patterns with the respective PXRD patterns reported in the CSD.
&#;1, being in accordance with previously reported values. In the case of PZQ cocrystals, carbonyl stretching ranged from to cm&#;1, and this downward shifting justified changes in H-bonding patterns in the resulting cocrystals. The C=O stretching vibrations of dicarboxylic acid coformers ranged from to cm&#;1, while in the cocrystals these vibrations ranged from to cm&#;1: the shifting of carbonyl stretching vibration to a larger wavenumber requires further energy, which is consistent with weaker intermolecular interactions (C-H···O and O-H···O) [Concerning FT-IR analyses, all the spectra of PZQ cocrystals were different from the starting materials. The two carbonyl amidic groups of pure PZQ were detected at and cm, being in accordance with previously reported values. In the case of PZQ cocrystals, carbonyl stretching ranged from to cm, and this downward shifting justified changes in H-bonding patterns in the resulting cocrystals. The C=O stretching vibrations of dicarboxylic acid coformers ranged from to cm, while in the cocrystals these vibrations ranged from to cm: the shifting of carbonyl stretching vibration to a larger wavenumber requires further energy, which is consistent with weaker intermolecular interactions (C-H···O and O-H···O) [ 58 ]. According to the thermal behavior, DSC and TGA analyses were both performed. No dehydration was seen in all cocrystals and cocrystal melting points were different from the starting materials. Precisely, melting points were detected at 159.9, 147.7, 141.2, 126.2, and 122.9 °C for PZQ-OXA, PZQ-MALO, PZQ-SUC, PZQ-GLU, and PZQ-ADI, respectively, showing a consistent effect between the spacer group of each coformer and the cocrystal melting point: the higher the number of carbons in the coformer space group, the lower the cocrystal melting event.
From the TGA results, it was evident that PZQ-OXA, PZQ-MALO, and PZQ-SUC started to dissociate at about 163, 147, and 167 °C, respectively, whereas PZQ-GLU and PZQ-ADI were thermally more stable and started to decay at 199 and 208 °C, respectively.
Passing to biopharmaceutics aspects, results from the solubility studies showed a substantial improvement of PZQ cocrystal aqueous solubility when compared to pure PZQ. Indeed, pure PZQ showed a solubility of 0.392 ± 0.1 mg/mL, in agreement with literature data [ 13 76 ], while the highest and lowest PZQ cocrystal solubilities observed were 10.5 ± 1.7 and 3.7 ± 1.08 mg/mL, respectively.
All cocrystals showed an improved dissolution profile with a descending order at 90 min as PZQ-SUC (68%), PZQ-ADI (57.96%), PZQ-GLU (54.05%), PZQ-OXA (51%), and PZQ-MALO (46.05%).
According to the pharmacokinetics results, an enhanced oral bioavailability in rabbits was observed for all cocrystals compared to pure PZQ (AUC 10.06 ± 2.51 μg h/mL, with PZQ-SUC exhibiting the highest oral bioavailability (33.84 ± 6.05 μg h/mL) and PZQ-MALO the lowest (15.40 ± 3.65 μg h/mL). Furthermore, the cocrystals Cmax reached in 1 h were higher compared to pristine PZQ, except for PZQ-OXA and PZQ-MALO.
The results of solubility, in vitro dissolution tests, and oral bioavailability were all consistent, as the enhanced solubility was translated into an improved dissolution and oral bioavailability. On the contrary, no consistency of the spacer group effect was seen in the biopharmaceutical properties and pharmacokinetics parameters.
Finally, PZQ cocrystals were also prepared via slurry in the presence of HPC to investigate the effect of polymer on their preparation. The results showed that HPC did not inhibit PZQ cocrystal formation except for PZQ-SUC, as PXRD analysis evidenced prominent peaks of residual PZQ in that sample.
w
/w
ratios were prepared, and, for comparison, the same inclusion experiments were carried out with pristine PZQ in the same silica material. N2 adsorption&#;desorption analysis, PXRD, IR spectroscopy, 13C SSNMR, DSC, and field-emission SEM (FE-SEM) were used to characterize the nanoconfined materials and compared to SB-15, raw PZQ, and pure PZQ-GLU cocrystal. N2 adsorption&#;desorption analysis showed a complete filling of the available channels of Sb-15 for the composition 50:50w
/w
and, therefore, the composites with 50:50w
/w
ratio of SBA-15/PZQ and SBA-15/PZQ-GLU were selected for further studies. FE-SEM images evidenced a rod-like morphology for SBA-15/PZQ-GLU instead of the smooth surface appearance of the micrometer-sized PZQ-GLU crystals. The PXRD pattern of SB-15/PZQ-GLU presented a broad halo, characteristic of raw amorphous SBA-15, with low-intensity diffraction peaks overlapping with the PZQ-GLU cocrystal, while SBA-15/PZQ exhibited the typical halo diffusion for amorphous material, and no peaks of crystalline PZQ were seen. As for the FT-IR results, the two C=O stretching vibrations of the GLU coformer at and cm&#;1 also remained in SBA-15/PZQ-GLU, supporting the cocrystal presence in the nanoconfined compound. For SBA-15/PZQ, the two C=O bands of raw PZQ ( and cm&#;1) were joined as one broad band at cm&#;1, behavior already observed by Perissutti and coworkers in amorphous dispersions [13C CPMAS and SPE (Single Pulse Excitation) allowed the conclusion that SBA-15/PZQ-GLU was a mixture of a cocrystalline bulk solid (outside the mesopores) and a more mobile phase (inside the mesopores), the latter being more evident in composites at lower ratios of PZQ-GLU. Through thermal analysis, SBA-15/PZQ did not show a distinct melting transition, confirming the amorphous state of the nanoconfined drug; SBA-15/PZQ-GLU presented two broad endothermic transitions at 63.0 and 117.2 °C, confirming the copresence of two types of phases: the cocrystalline bulk solid outside the mesopores was detected at 117.2 °C, like pure PZQ-GLU melting at 123.0 °C, and the more mobile phase inside the mesopores at 63.0 °C, as nanoconfined drugs are known to display a huge depression of their melting temperature [118,GLU···O=CPZQ intermolecular H-bonds in the cocrystal in comparison to C-HPZQ···O=CPZQ contacts in (RS)-PZQ and to a larger crystal lattice volume of PZQ over the PZQ-GLU cocrystal (.1 vs. .9 Å3).Recently, Salas-Zúñiga and coauthors combined the potential of the previously obtained PZQ cocrystals with size reduction by confinement in mesoporous silica material (i.e., SB-15) with nanopores (ca. 5.6 nm pore size) to further improve the solubility and dissolution of the materials [ 98 ]. Among the seven previously solved new cocrystals, PZQ-GLU was chosen for the following reasons: (i) it had a smaller unit cell volume than racemic PZQ; (ii) it melted at a lower temperature than raw (RS)-PZQ (ΔT = &#;15.8 °C), allowing it to perform the melt loading method for confinement; (iii) its recrystallization from the melt gave only the original cocrystal and displayed a unique fingerprint in the IR spectrum, thus making it easily recognizable. Four SBA-15/cocrystalratios were prepared, and, for comparison, the same inclusion experiments were carried out with pristine PZQ in the same silica material. Nadsorption&#;desorption analysis, PXRD, IR spectroscopy,C SSNMR, DSC, and field-emission SEM (FE-SEM) were used to characterize the nanoconfined materials and compared to SB-15, raw PZQ, and pure PZQ-GLU cocrystal. Nadsorption&#;desorption analysis showed a complete filling of the available channels of Sb-15 for the composition 50:50and, therefore, the composites with 50:50ratio of SBA-15/PZQ and SBA-15/PZQ-GLU were selected for further studies. FE-SEM images evidenced a rod-like morphology for SBA-15/PZQ-GLU instead of the smooth surface appearance of the micrometer-sized PZQ-GLU crystals. The PXRD pattern of SB-15/PZQ-GLU presented a broad halo, characteristic of raw amorphous SBA-15, with low-intensity diffraction peaks overlapping with the PZQ-GLU cocrystal, while SBA-15/PZQ exhibited the typical halo diffusion for amorphous material, and no peaks of crystalline PZQ were seen. As for the FT-IR results, the two C=O stretching vibrations of the GLU coformer at and cmalso remained in SBA-15/PZQ-GLU, supporting the cocrystal presence in the nanoconfined compound. For SBA-15/PZQ, the two C=O bands of raw PZQ ( and cm) were joined as one broad band at cm, behavior already observed by Perissutti and coworkers in amorphous dispersions [ 50 ]. Further, a comparison ofC CPMAS and SPE (Single Pulse Excitation) allowed the conclusion that SBA-15/PZQ-GLU was a mixture of a cocrystalline bulk solid (outside the mesopores) and a more mobile phase (inside the mesopores), the latter being more evident in composites at lower ratios of PZQ-GLU. Through thermal analysis, SBA-15/PZQ did not show a distinct melting transition, confirming the amorphous state of the nanoconfined drug; SBA-15/PZQ-GLU presented two broad endothermic transitions at 63.0 and 117.2 °C, confirming the copresence of two types of phases: the cocrystalline bulk solid outside the mesopores was detected at 117.2 °C, like pure PZQ-GLU melting at 123.0 °C, and the more mobile phase inside the mesopores at 63.0 °C, as nanoconfined drugs are known to display a huge depression of their melting temperature [ 117 119 ]. To summarize, several characterization techniques demonstrated that racemic PZQ was loaded mainly in amorphous form, whereas a more mobile/solid-like phase of PZQ-GLU was nanoconfined within SBA-15. The contrasting behavior of the two nanoconfined solids was attributed to the more robust O-H···O=Cintermolecular H-bonds in the cocrystal in comparison to C-H···O=Ccontacts in (RS)-PZQ and to a larger crystal lattice volume of PZQ over the PZQ-GLU cocrystal (.1 vs. .9 Å).
0&#;90min up to 5.1-fold in comparison to pristine PZQ, similarly to SBA-15/PZQ. Under the second condition, the nanoconfined composite generated an immediate release of the drug. Thus, this study confirmed that nanoconfinement combined with cocrystallization is an efficient tool to further improve some properties of poorly soluble drugs like PZQ.The impact of pure PZQ and the cocrystal confined in SBA-15 was further examined by performing dissolution studies in two different experimental settings: (i) under non-sink conditions with a saturation index (SI) [ 120 ] of 0.014 for PZQ and 0.02 for PZQ-GLU, in the presence of a precipitation inhibitor polymer (Methocel 60 HG), and (ii) under non-sink conditions with an SI of 1.99 for PZQ and PZQ-GLU in aqueous HCl pH 1.2 at 37 °C. In the first case, SBA-15/PZQ-GLU showed a sustained solubilization of PZQ, increasing the AUCup to 5.1-fold in comparison to pristine PZQ, similarly to SBA-15/PZQ. Under the second condition, the nanoconfined composite generated an immediate release of the drug. Thus, this study confirmed that nanoconfinement combined with cocrystallization is an efficient tool to further improve some properties of poorly soluble drugs like PZQ.
1), confirming the enantiomerically pure nature of PZQ. Structural analysis revealed that the dominant supramolecular interactions in the new crystals were quite similar: both amide groups of (R)-PZQ were involved in heterosynthons consisting of O-Hcoformer···O=CPZQ H-bonds with the COOH groups of the dicarboxylic acid. Also, complementary secondary (N)C-HPZQ···O=Ccoformer interactions generated two slightly distinct eight- and nine-membered cyclic double bridged heterodimeric synthons.Finally, focusing the attention, once again, on PZQ-GLU plus also PZQ-SUC cocrystals, the same scientific group reported two novel cocrystals obtained through LAG in the presence of the above-mentioned coformers ground with enantiomerically pure (R)-PZQ in a 1:1 molar ratio [ 67 ]. The new cocrystals (i.e., (R)-PZQ-GLU and (R)-PZQ-SUC) were deeply characterized and compared with previously reported analogous cocrystals obtained with racemic PZQ (hereinafter referred as (RS)-PZQ-GLU and (RS)-PZQ-SUC), pure (R)-enantiomer hemihydrate (i.e., (R)-PZQ-HH), and pristine (RS)-PZQ. PXRD patterns of the new phases clearly differed from both starting materials and (RS)-PZQ-GLU and (RS)-PZQ-SUC. Suitable single crystals for SXRD analysis were obtained through classical solution crystallization and their simulated PXRD patterns agreed with experimental ones. (R)-PZQ-GLU and (R)-PZQ-SUC (indexed as KEQVEL and KEQVAH in the CSD, respectively) crystallize in a chiral space group (P2), confirming the enantiomerically pure nature of PZQ. Structural analysis revealed that the dominant supramolecular interactions in the new crystals were quite similar: both amide groups of (R)-PZQ were involved in heterosynthons consisting of O-H···O=CH-bonds with the COOH groups of the dicarboxylic acid. Also, complementary secondary (N)C-H···O=Cinteractions generated two slightly distinct eight- and nine-membered cyclic double bridged heterodimeric synthons.
In the IR spectra of (R)-PZQ-GLU and (R)-PZQ-SUC, the band observed at roughly cm&#;1 for the H2O molecules in starting (R)-PZQ-HH was missing, highlighting the anhydrous nature of the two cocrystals. Moreover, C=O stretching vibrations of PZQ were displaced at smaller wavenumbers, while C=O bands of coformers were displaced at larger wavenumbers: these shifts suggested that H-bond interactions in the cocrystals were stronger than those observed between H2O molecules and (R)-PZQ in the hemihydrate and those of dicarboxylic acid dimers.
The thermal behavior of the two cocrystals was slightly different: (R)-PZQ-SUC presented a melting point at 130.2 °C, which was in between the starting materials (112.9 °C for (R)-PZQ-HH and 184.9 °C for SUC), whereas (R)-PZQ-GLU exhibited a melting temperature of 81.0 °C, being lower than the individual components (112.9 °C for (R)-PZQ-HH and 102.9 °C for GLU). This behavior could find a possible explanation in the difference of stability, solubility, and the dissolution rate of the two systems. Therefore, the authors performed IDR and powder dissolution studies on the two enantiomerically pure cocrystals and compared them to the corresponding racemic (RS)-PZQ-GLU and (RS)-PZQ-SUC, pristine (R)-PZQ-HH, and (RS)-PZQ in a simulated gastric dissolution medium (HCl, pH 1.2, 37 °C) where dicarboxylic acids were fully protonated. IDR results showed that all four cocrystals (both enantiomerically pure and racemic) had superior dissolution compared to (RS)-PZQ and (R)-PZQ-HH, with advantages by factors ranging from two to five, depending on the cocrystal, with respect to (R)-PZQ-HH. The IDRs were in the order (R)-PZQ-GLU > (RS)-PZQ-SUC &#; (RS)-PZQ-GLU > (R)-PZQ-SUC. Interestingly, the IDRs of the four cocrystals exhibited a slope that changed significantly in the range of 30&#;60 min, which was attributed to a phase transformation into (R)-PZQ-HH and (RS)-PZQ-HH, as attested to by IR and PXRD analyses of the IDR tablets. In powder dissolution tests, these studies were performed in non-sink conditions by adding dissolution medium, simulating the gastric fluid to an excess of cocrystal, to assess the supersaturation potential of the enantiomerically pure cocrystals and their racemic counterparts. In this case, (R)-PZQ-GLU, (RS)-PZQ-GLU, and (RS)-PZQ-SUC showed increased solubility compared to (RS)-PZQ and (R)-PZQ-HH, giving maximum concentrations of 4.6 mM, 3.8 mM, and 2.7 mM, respectively, within the first 5 min. On the contrary, for the enantiomerically pure (R)-PZQ-SUC cocrystal, the dissolution profile was like that of (R)-PZQ-HH, suggesting no generation of a supersaturated solution.
After a period of approximately 5 min, the concentration of the three cocrystals decayed to a concentration like (RS)-PZQ and (R)-PZQ-HH, indicating a rapid reprecipitation. PXRD analysis on the recovered samples suggested a conversion of the cocrystals to the parent compound (RS)-PZQ and (R)-PZQ-HH, as in the case of IDR.
Coming back to the cocrystallization strategy, three years after the discovery of PZQ cocrystals with dicarboxylic acids [ 58 ], Sánchez-Guadarrama et al. used cocrystallization to achieve a chiral resolution of racemic PZQ via the formation of cocrystal diastereoisomers with L-Malic Acid (L-MAL), as discussed in detail in paragraph 3. In short, the authors performed LAG with 10 μL of AcT using a 1:1 stoichiometric mixture of (RS)-PZQ and L-MAL for 30 min at 25 Hz and obtained two enantiomerically pure cocrystals (i.e., (R)-PZQ/L-MAL and (S)-PZQ/L-MAL), the structures of which were solved and deposited in the CSD [ 68 ].
&#;1 were downward shifted and only one broader band was noticed at , , and cm&#;1 for PZQ-MAL, PZQ-SA, and PZQ-TA, respectively, suggesting H-bond interactions between PZQ and cocrystal formers and an involvement of both carbonyl groups in H-bond formation. Additionally, the shift of band from to cm&#;1, assigned to C=O stretching vibrations of the carboxylic group of SA, confirmed the formation of the H-bond between PZQ carbonyl groups and the SA carboxylic group. Passing to MAL, considerable differences in band intensities at and cm&#;1 characteristic for -OH and C=O of the coformer could be noticed, indicating that the hydroxyl group of MAL probably participated in the H-bonds with PZQ carbonyl groups. In the case of TA, bands at and cm&#;1, assigned to the stretching of the alcoholic group of TA, resulted in a shift in PZQ-TA, and a new band around cm&#;1 also emerged, confirming the interaction with PZQ carbonyl groups through the TA alcoholic group. Worthy of notice is the case of PZQ-CA, in which for the FT-IR spectrum no shifts of characteristic bands of PZQ were detected, while a shift of the C=O group of the coformer was noticed from , , and cm&#;1 to , , and cm&#;1 that anyway suggested the formation of a new solid form. All the prepared cocrystals showed pH-dependent solubility, with the highest saturation solubility observed at pH 4.5, since the ionizable groups of coformers were all ionized at that value, as evident from their pKa [Similarly, Cugovčan and coworkers reported having obtained PZQ cocrystals with citric (CA), malic (MAL), salicylic (SA), and tartaric acids (TA) by means of LAG [ 39 ]. Precisely, (RS)-PZQ was milled in the presence of each coformer in a 1:1 stoichiometry for 30 min at 25 Hz with or without an addition of 15 μL of absolute EtOH, LAG being the most efficient technique. PXRD patterns of PZQ-CA, PZQ-MAL, PZQ-TA, and PZQ-SA showed several new peaks and no residual peaks of the starting materials, confirming the cocrystal formation in a 1:1 molar ratio. DSC analyses revealed a new endothermic peak with an onset of 51.07 °C for PZQ-CA and 150.81 °C for PZQ-TA, whereas DSC was not useful to elucidate melting events in the cases of PZQ-MAL and PZQ-SA. Further information about the cocrystal formation was, instead, obtained by FT-IR analysis. The two typical carbonyl stretching vibrations of PZQ Form A observed at and cmwere downward shifted and only one broader band was noticed at , , and cmfor PZQ-MAL, PZQ-SA, and PZQ-TA, respectively, suggesting H-bond interactions between PZQ and cocrystal formers and an involvement of both carbonyl groups in H-bond formation. Additionally, the shift of band from to cm, assigned to C=O stretching vibrations of the carboxylic group of SA, confirmed the formation of the H-bond between PZQ carbonyl groups and the SA carboxylic group. Passing to MAL, considerable differences in band intensities at and cmcharacteristic for -OH and C=O of the coformer could be noticed, indicating that the hydroxyl group of MAL probably participated in the H-bonds with PZQ carbonyl groups. In the case of TA, bands at and cm, assigned to the stretching of the alcoholic group of TA, resulted in a shift in PZQ-TA, and a new band around cmalso emerged, confirming the interaction with PZQ carbonyl groups through the TA alcoholic group. Worthy of notice is the case of PZQ-CA, in which for the FT-IR spectrum no shifts of characteristic bands of PZQ were detected, while a shift of the C=O group of the coformer was noticed from , , and cmto , , and cmthat anyway suggested the formation of a new solid form. All the prepared cocrystals showed pH-dependent solubility, with the highest saturation solubility observed at pH 4.5, since the ionizable groups of coformers were all ionized at that value, as evident from their pKa [ 121 122 ]. Among the four cocrystals, the PZQ-MAL cocrystal showed the highest solubility. Considering the dissolution studies, all of the new systems presented significantly superior dissolution properties compared to those of the pure drug. Interestingly, the authors noticed that PZQ-MAL was chemically unstable and favored PZQ photodegradation. Despite these characterizations, no new crystal structure was solved and reported in the CSD.
Subsequently, Yang et al. published a work in which conformations of PZQ and three flavonols used as coformers, namely kaempferol (KAE), quercetin (QUE), and myricetin (MYR), were analyzed through theoretical calculations to predict cocrystal formation [ 69 ]. They based their study on molecular electrostatic potential surfaces (MEPS) using density functional theory (DFT), which can accurately reflect changes in the intermolecular interaction sites caused by the conformational changes. This system provides a useful method for predicting the interactions of the participant molecules in a cocrystal [ 123 ]. Precisely, they started from the determination of the conformations of flavonols and PZQ Form A by means of DFT and their Boltzmann distributions at 300 K. Then, MEPS for each conformation were calculated to predict the difference in the interaction site pairing energies (ΔE) and, therefore, the cocrystal formation. As is well known from the literature, the smaller the ΔE value, the greater the probability of forming cocrystals [ 123 ].
KAE, QUE, and MYR exhibited two main conformations. For KAE and MYR, the difference among them was very small; for QUE, the two conformations differed more greatly due to the asymmetric distribution of the hydroxyl groups: according to the CCDC, conformation 1 (QUE C1) accounts for approximately 75% and conformation 2 (QUE C2) for roughly 25%. PZQ showed four main conformations due to the rotation of the cyclohexylcarbonyl, showing two of them with the carbonyl groups on the opposite site (
anti
conformation) (i.e., C1 and C2) and the other two with the carbonyl groups on the same site (syn
conformation) (i.e., C3 and C4). The CCDC reports a predominancy of conformations C1 and C3 for PZQ. ΔE values, calculated considering the effective number of H-bond acceptors and donors of PZQ and flavonols, enabled the prediction of four different cocrystals, namely PZQ-KAE, PZQ-QUE 1, PZQ-QUE 2, and PZQ-MYR. For KAE, the two maxima of the MEPS were located on the region of the hydroxyl groups which could form H-bonds, with the two minima of MEPS on PZQ C1. Considering that the two hydroxyl sites were located on both sides of the KAE molecule, the cocrystal was speculated to be composed of a stoichiometric ratio of 2:1 PZQ-KAE. For QUE C1, there were two pairs of maxima of the MEPS located on the hydroxyl groups that could interact with the two minima of MEPS on PZQ. For QUE C2, the two maxima sites of MEPS located in the region of the hydroxyl groups could only interact with the minima of MEPS on PZQ C3. For MYR, the situation was the same observed for QUE C2: the interaction was supposed to be only with PZQ in conformation C3.All four predicted cocrystals were obtained experimentally by means of the suspension-stirring method ( Figure 9 ) and characterized through PXRD. For KAE, the PZQ-KAE cocrystal was obtained in a 2:1 stoichiometry. For QUE, two kinds of cocrystals were achieved as assessed by PXRD. The stoichiometric ratio of one was 2:1 and the single crystal, named PZQ-QUE 1, was also grown. The stoichiometric ratio of the other cocrystal, PZQ-QUE 2, was 1:1, but no single crystal was obtained in that case. For MYR, the cocrystal PZQ-MYR showed a 1:1 stoichiometric ratio and the single crystal was also grown.
The three single crystals were all block-shaped crystals and their structural analysis by SXRD clarified the type of interactions of the molecules in the cocrystal packing. PZQ-KAE (indexed as ANAYOG) crystallizes in the monoclinic space group P21/
a
and has four formula units per unit cell (Z = 4). The asymmetric unit contains one KAE and two PZQ molecules, consistent with theoretical calculations, with the presence of PZQ conformation C1 (anti
-conformation). PZQ-QUE 1 (ANAYUM in the CSD) crystallizes in the triclinic space group P-1 and shows two formula units per unit cell (Z = 2). The asymmetric unit contains one QUE and two PZQ molecules with both PZQ conformations C1 and C3, thus presenting one molecule inanti
and the other insyn,
both interacting with QUE. PZQ-MYR (ANAZAT in the CSD) crystallizes in the monoclinic space group P21/c
and has four formula units per unit cell (Z = 4). The asymmetric unit contains one MYR and one PZQ molecule, consistent with the 1:1 stoichiometry, with the presence of PZQ conformation C3 (syn
conformation). Therefore, the SXRD results were demonstrated to be consistent with the predicted interactions.Experimentally obtained cocrystals were also characterized through DSC and FT-IR spectroscopy. All four cocrystals presented a single melting peak which was in between the starting materials, with increasing values in the order PZQ-KAE < PZQ-QUE 1 < PZQ-QUE 2 < PZQ-MYR. Considering instead the FT-IR analysis, the main differences were observed in the wavenumbers above cm&#;1: raw flavonols contain in their structures crystal water which interacts with their hydroxyl groups; after the formation of cocrystals with PZQ, no crystal water exists due to the interaction between flavonol hydroxyl groups and PZQ carbonyl groups, and therefore peaks in the region of cm&#;1 become weak.
Finally, as both flavonols and PZQ belong to BCS class II, flavonols being less soluble than PZQ, solubility studies were also carried out. The results showed that the solubility of the compound with higher solubility (i.e., PZQ) was reduced after the formation of the cocrystals, while the solubilities of the compounds with poor solubility (i.e., flavonols) were increased approximately three-fold.
anti
-conformation with the carbonyl groups in opposite directions and&#;in some cases&#;with a 90° rotation of the cyclohexyl ring. Moreover, all the crystal structures were centrosymmetric, containing therefore both the (R)- and the (S)-enantiomer of PZQ. Based on the intermolecular interaction patterns and the packing of PZQ enantiomers, the 12 crystal structures were organized into four different classes. The first class included PZQ-2,5-DHA, PZQ-2,5-DHA ACN, PZQ-4-HA, and PZQ-4-ASA ACN (refcodes in the CSD AVEHUH, AVEHIV, AVEJIX, and AVEJOD, respectively), which were characterized by one-dimensional enantiopure chains where both carbonyl groups of PZQ interacted with the coformer through H-bonds. As the crystal structures are centrosymmetric, they also contain chains of opposite chirality. The two cocrystal solvates of the class were isostructural, and their enantiopure chains lied in the same crystallographic direction ([Using the knowledge gained from the previous eight PZQ cocrystal structures deposited in the CSD, Devogelaer and coauthors used a network-based link-prediction algorithm [ 124 125 ] to predict 30 new coformer candidates for PZQ [ 70 ]. The list included very different compounds, such as five aliphatic dicarboxylic acids (four of which were previously considered by Espinosa-Lara et al.), benzoic acids derivatives, and aromatic compounds with hydroxyl, amine, and nitro groups. Also, salicylic acid and an enantiomer of tartaric acid were present in the list, the crystal structures of which were not previously solved [ 39 ]. Interestingly, 1,2,4,5-tetrafluoro-3,6-di-iodobenzene (DIFTB) was also predicted as a possible coformer for PZQ, even though its structure is very dissimilar to the other expected and reported coformers for PZQ. From these 30 coformers, 17 experimental indications for cocrystals were obtained but only 12 out of 17 were successfully grown as single crystals and analyzed through SXRD. Specifically, eight cocrystals were binary systems (i.e., PZQ-1,2,4,5-tetrafluoro-3,6-di-iodobenzene (PZQ-DITFB), PZQ-4-hydroxybenzoic acid (PZQ-4-HA), PZQ-3,5-dinitrobenzoic acid (PZQ-3,5-DNA), PZQ-hydroquinone (PZQ-HQ), PZQ-vanillic acid (PZQ-VA), PZQ-2,5-dihydroxybenzoic acid (PZQ-2,5-DHA), PZQ-2,4-hydroxybenzoic acid (PZQ-2,4-HA), and PZQ-orcinol (PZQ-ORC)), and four were cocrystal solvates (i.e., PZQ-salicylic acid hydrate (PZQ-SA MH), PZQ-4-aminosalicylic acid acetonitrile solvate (PZQ-4-ASA ACN), PZQ-2,5-dihydroxybenzoic acid acetonitrile solvate (PZQ-2,5-DHA ACN), and PZQ-3,5-dihydroxybenzoic acid acetonitrile solvate (PZQ-3,5-DHA ACN)). In all 12 crystal structures, PZQ molecules displayed the-conformation with the carbonyl groups in opposite directions and&#;in some cases&#;with a 90° rotation of the cyclohexyl ring. Moreover, all the crystal structures were centrosymmetric, containing therefore both the (R)- and the (S)-enantiomer of PZQ. Based on the intermolecular interaction patterns and the packing of PZQ enantiomers, the 12 crystal structures were organized into four different classes. The first class included PZQ-2,5-DHA, PZQ-2,5-DHA ACN, PZQ-4-HA, and PZQ-4-ASA ACN (refcodes in the CSD AVEHUH, AVEHIV, AVEJIX, and AVEJOD, respectively), which were characterized by one-dimensional enantiopure chains where both carbonyl groups of PZQ interacted with the coformer through H-bonds. As the crystal structures are centrosymmetric, they also contain chains of opposite chirality. The two cocrystal solvates of the class were isostructural, and their enantiopure chains lied in the same crystallographic direction ([ 111 ]). In case of PZQ-ORC (indexed as AVEHOB in the CSD), zigzag chains were visible and chains with an equal chirality stacked on top of each other. The second class comprised PZQ-HQ, PZQ-2,4-HA, and PZQ-3,5-DHA ACN (refcodes AVEKIJ, AVEJUJ, and AVEKEU). In this case, H-bonding patterns induced the formation of chains containing both the enantiomers of PZQ (1:1 molar ratio) and the coformer. Like the first class, both carbonyl groups of the alternating PZQ enantiomers took part in H-bond interaction with the coformer. On the contrary, for PZQ-VA, PZQ-3,5-DNA, and PZQ-SA MH (refcodes AVEJAP, AVEJET, and AVEHER), a class of so-called racemic pair cocrystals was identified where the (R)- and (S)-enantiomers of PZQ interact via H-bonds similarly to PZQ polymorph B. The fourth class just enclosed PZQ-DITFB (indexed as AVEKAQ in the CSD), which was the only molecule for which H-bonds were precluded, leaving halogen bonds and π&#;π interactions as plausible alternatives for cocrystal formation. Specifically, alternating PZQ enantiomers interact via carbonyl&#;iodide interactions, with the coformer and the fluorine atoms of the coformers additionally interacting with the hydrogens of the aromatic and cyclohexyl rings of PZQ.
In a subsequent work, the same group focused attention on conducting a thorough comparison of the results obtained from the screening methods LAG, solvent evaporation (SE), and saturation temperature measurements (STM) ( Figure 9 ) to review their advantages and drawbacks in cocrystal preparation [ 126 ]. LAG, SE, and STM enabled the identification of the previously discussed 17 cocrystals, with 14 showing stability and 12 new crystal structures solved. Going into detail, LAG experiments were performed by grinding a 1:1 molar ratio of PZQ and each coformer in the presence of reagent grade MeOH, EtOH, isopropanol (IPOH), AcT, and EA, and the output of the experiments was assessed by means of PXRD analysis. With LAG experiments, 11 coformers out of 30, namely 3,5-DNA, SA, DITFB, 4-HA, 4-ASA, HQ, VA, 2,5-DHA, 3,5-DNA, 2,4-DHA, and ORC, gave a new pattern not presenting traces of the starting materials. In most cases, systems screened with LAG in multiple solvents resulted in the same solid phase formation. Two coformers, i.e., VA and 2,5-DHA, gave, instead, two different PXRD patterns, depending on the solvent used. Therefore, 13 new systems for 11 positive coformers were identified through LAG experiments. Clarifications about the nature of the systems (i.e., cocrystals, cocrystal solvates, or cocrystal polymorphs) and their stoichiometry were given by the SXRD analysis of the crystal structures. Among the 13 new PXRD patterns, single-crystal growth experiments confirmed 12 new cocrystal structures where the simulated patterns corresponded to those obtained experimentally. Eight coformers gave 1:1 cocrystals with PZQ: DITFB, 4-HA, 4-ASA, HQ, VA, 2,5-DHA, 2,4-DHA, and ORC. Four cocrystals out of twelve were cocrystal solvates, with three of these being solvates with ACN (i.e., 4-ASA, 2,5-DHA, and 3,5-DHA). The fourth was a cocrystal hydrate unexpectedly obtained with SA through LAG in the presence of AcT, where water molecules were an impurity kept from ambient humidity. Interestingly, VA remained the unique semi-unclarified coformer: as above-mentioned, VA gave two different PXRD patterns if either using EtOH or ACN through LAG. The phase produced by using EtOH was a 1:1 cocrystal, whose structure was solved by SXRD. The other phase formed with ACN was not obtained through single-crystal growth experiments and therefore remained a question mark.
SE experiments were performed with the same solvents of LAG to make comparison consistent. Ten coformers out of thirty (i.e., 3,5-DNA, PIM, SA, DITFB, 4-HA, HQ, VA, 2,5-DHA, 3,5-DHA, and 2,4-DHA) gave a new pattern not presenting traces of starting materials. Even in case of SE, 12 new PXRD patterns for 10 positive coformers were identified, and, as LAG outcomes, VA and 2,5-DHA gave two different PXRD patterns, depending on the solvent used. Worthy of notice is the fact that PXRD patterns obtained for HQ and 2,4-DHA through SE were not the same 1:1 cocrystals obtained by means of LAG, for which single crystals were grown. On the contrary, no single crystals were obtained for SE phases and the same problem was also observed for PIM, even though a new PXRD pattern was identified through SE experiments. For the other coformers, the PXRD patterns of SE products corresponded to those of LAG, except for 4-ASA, which did not give any cocrystal by means of SE, differing from LAG.
For STM, 9 coformers out of 30 (i.e., 3,5-DNA, DITFB, 4-HA, 4-ASA, HQ, VA, 2,5-DHA, 3,5-DHA, and 2,4-DHA) showed a positive response in cocrystallization. A false positive was observed for benzoic acid in EtOH, as PXRD confirmed a physical mixture of PZQ and benzoic acid. In total, 12 new PXRD patterns for 9 positive coformers were obtained. As for LAG and SE, VA and 2,5-DHA gave two different outcomes due to the solvent used. The same was observed for 2,4-DHA, whose new pattern obtained in EtOH was specific to STM. However, no single crystal was grown in this case, so it was unclear whether it was a cocrystal, a polymorph, or a cocrystal solvate. For all other positive coformers, PXRD patterns were consistent with those obtained by LAG.
To summarize, 12 new cocrystals were analyzed by means of SXRD and deposited in the CSD. All of them were identified with LAG, being the one found for ORC specific to LAG. For the VA coformer, two different patterns were obtained by using LAG, SE, and STM. The first was consistent with a 1:1 cocrystal, the crystal structure of which was solved through PXRD; the second remained an unresolved new phase, even though the possibility of a cocrystal solvate was excluded as the same result was obtained in several solvents. Presumably, this questioned phase could have been a cocrystal with a stoichiometry of 2:1 VA:PZQ, as the STM method required an excess of VA to obtain the experiments. Therefore, 13 stable cocrystals were discovered through LAG. With SE, 12 new systems were identified, with 9 in common with LAG and STM. The other three were specific to SE (PIM, HQ, and 2,4-DHA) and considered metastable due to inconsistency with LAG and STM experiments in the same conditions. As for STM, 12 new PXRD patterns were noticed, with 11 in common with LAG. A second cocrystal was obtained for 2,4-DHA in EtOH, being a specific result of STM.
In summary, LAG was identified as the best, quickest, and most efficient screening route for cocrystallization, followed by STM and SE.
In the same year, Liu and coworkers reported a work aiming at investigating the solubility of four PZQ-carboxylic acids cocrystals and understanding their structural features by exploring the intermolecular weak interactions through theoretical calculations [ 71 ]. Citric acid (CA), phtalic acid (PA), 3-hydroxybenzoic acid (3-HA), and 4-hydroxybenzoic acid (4-HA) were chosen as coformers due to their good solubility and ground with PZQ by means of LAG to achieve cocrystallization (i.e., PZQ-CA, PZQ-PA, PZQ-3-HA, and PZQ-4-HA) ( Figure 9 ). PZQ-CA was previously obtained, but its crystal structure was not solved [ 39 ]; the single crystal structure for PZQ-4-HA was obtained in a previous article but other characterization and solubility experiments were not performed [ 70 ]; PZQ-PA and PZQ-3-HA were previously predicted to form [ 70 ] but have never been obtained before.
The four cocrystals were fully characterized and the interactions between PZQ and each coformer were analyzed through theoretical calculations, namely atoms in molecules (AIM) topology analysis, electron density difference analysis (EDD), and energy decomposition analysis (EDA).
PXRD patterns of the new products showed characteristic reflections not attributable to the starting materials, suggesting a 1:1 stoichiometry for PZQ-CA, PZQ-PA, and PZQ-4-HA, whereas a 2:1 molar ratio was suggested for PZQ-3-HA. Stoichiometries were confirmed by means of SXRD through which the crystal structure solution was possible. Precisely, PZQ-CA (refcode DAJYUM in the CSD) crystallizes in a 1:1 molar ratio in the orthorhombic space group of P; PZQ-PA (refcode DAJZIB) in the P21/
c
space group of the monoclinic system; PZQ-3-HA and PZQ-4-HA (indexed as DAJZEX and AVEJIX01, respectively) crystallize in the triclinic space group of P-1. In all systems, PZQ carbonyl groups displayed ananti
-conformation and SXRD analysis revealed that the main interactions between PZQ and coformers were H-bonds between PZQ carbonyl groups and coformer hydroxyl groups in different interaction modes. This evidence was also confirmed by FT-IR results in which shifts at lower frequencies or the disappearance of -OH stretching bands of coformers and shifts at higher frequencies for C=O stretching of PZQ were noticed. DSC analysis revealed the melting points for the four systems: the melting peak of PZQ-CA was detected at 137.2 °C, being lower than those of the starting materials and thus suggesting a worse thermal stability; the same was observed for PZQ-3-HA, which showed a melting point at 109.3 °C. On the contrary, PZQ-PA and PZQ-4-HA presented melting events at 150.0 °C and 155.0 °C, respectively, being in between the two pure components and demonstrating a better thermal stability compared to pure PZQ.Theoretical calculation methods such as AIM and EDD revealed the existence of classical and nonclassical H-bond interactions: classical O-H···O H-bond interactions were confirmed to be present in the structures, but also nonclassical interactions such as C-H···O were found, even if their strength was lower based on the EDD. EDA was then used to determine all the minor interactions present in the four systems and to clarify which of them mainly contributed to the formation of the cocrystal: H-bond interactions were demonstrated to be the main contributor, whereas forces such as dispersion and induction were relatively minor even though they could not be completely ignored.
Furthermore, in vitro solubility tests in four media with different pH values were carried out and the results showed improved solubilities of all the cocrystals compared to pristine PZQ: after 4 h, the cocrystals&#; solubility was about 4 times higher in the pH 1.2 medium, roughly 2 times higher in the pH 4.5 medium and water, and about 3.3 times higher in the pH 6.8 medium. PZQ-CA solubility was the worst of the four systems.
In , the same scientific group also reported PZQ cocrystallization in the presence of polyhydroxy phenolic acids, namely protocathecuic acid (PA), gallic acid (GA), and ferulic acid (FA) [ 72 ]. They prepared five different cocrystals through LAG and SE methods: precisely, PZQ-PA, PZQ-GA, and PZQ-FA were obtained by grinding in a mortar a 1:1 molar ratio of PZQ and each coformer in the presence of a certain amount of ACN ( Figure 9 ); SE, performed with the same solvent, facilitated obtaining two cocrystal solvates, i.e., PZQ-PA-ACN and PZQ-GA-ACN, plus the same PZQ-FA cocrystal of LAG. These five new cocrystals were deeply characterized at the solid state, and the crystal structures of PZQ-PA-CAN, PZQ-GA-ACN, and PZQ-FA were solved.
Unique endothermic peaks were visible in DSC curves for the three anhydrous cocrystals, and their melting points were in between those of PZQ and each coformer; the two cocrystal solvates, instead, showed endothermic events at 86.16 °C and 104.88 °C for PZQ-PA-ACN and PZQ-GA-ACN, respectively, corresponding to desolvation, as attested to also by TGA results. No further endothermic events were observed after desolvation, attesting that the crystalline lattice collapses after the loss of crystal ACN.
PXRD analyses of the five cocrystals showed different patterns compared to those of PZQ and the coformers. Interestingly, the PZQ-PA and PZQ-GA patterns were similar, and the same behavior was observed also for PZQ-PA-ACN and PZQ-GA-ACN: the explanation lies in the very similar PA and GA structures that only differ in the five-position hydroxyl group.
As the SE method enabled access to single crystals, three out of five cocrystals were analyzed by SXRD and their crystal structures were deposited in the CSD with deposition numbers of , , and for PZQ-PA-ACN, PZQ-GA-ACN, and PZQ-FA, respectively. Both cocrystal solvates belong to the monoclinic I2/
a
space group and their ASU contains one PZQ, one PA/GA, and one solvent molecule, confirming a 1:1:1 PZQ:coformer:solvent stoichiometry. PZQ-FA belongs, instead, to the monoclinic space group of P21/n
and its ASU presents one PZQ and one FA molecule. In the two cocrystal solvates, phenolic acid forms H-bonds with PZQ and the solvent in the form of a monomer, while in PZQ-FA, phenolic acid first forms dimer and then interacts with PZQ by H-bonding. All three cocrystals displayed ananti
-conformation of carbonyls of PZQ molecules.Also, FT-IR analyses confirmed H-bond interactions and therefore cocrystal formation. Precisely, the interaction was assessed by the C=O stretching shift of carboxylic acids: in the case of cocrystal solvates, the characteristic vibration of the conjugated carboxylic acid monomer was found at cm&#;1, while the characteristic absorption peaks belonging to C=O stretching vibration of conjugated carboxylic acid dimer were observed at , , and cm&#;1 for PZQ-PA, PZQ-GA, and PZQ-FA, respectively.
The mechanism of cocrystal formation was also discussed via theoretical calculations, including molecular interaction energy, EDD, and MEPS.
Regarding the interaction energies of the cocrystal solvates, PZQ-PA-ACN exhibited lower bonding energy than PZQ-GA-ACN and the same was observed for the energies between PA and ACN and GA and ACN, supporting the much lower desolvation temperature of PZQ-PA-ACN compared to PZQ-GA-ACN. In the PZQ-FA, the interaction energy was the lowest, attesting that the connection between the FA dimer was the most stable and easy to generate.
EDD and MEPS highlighted a decreasing electron density around hydroxyl oxygen atoms of coformers and an increasing electron density around the carbonyl oxygen of PZQ and, in case of cocrystal solvates, cyano nitrogen atoms of ACN, confirming the formation of H-bonds.
Passing to biopharmaceutic properties, anhydrous cocrystals were selected to carry out solubility evaluations. Four different media (i.e., aqueous HCl solution (pH = 1.2), acetate buffer (pH = 4.5), phosphate buffer (pH = 6.8), and water) were used. Except for PZQ-PA in acetate buffer, the dissolution results in the four media differ remarkably from that of PZQ, and the cocrystals&#; solubility was better. Based on the improvement of the solubility, PZQ-FA was selected as representative to also evaluate the biological activity in vivo. The Tmax and Cmax of PZQ and PZQ-FA were basically the same, but the absorption degree of the latter was superior.
71,71,Recently, Yang et al. reported another work with the application of artificial intelligence in cocrystal screening [ 127 ]. Precisely, they provided a data-driven cocrystal prediction method based on the eXtreme Gradient Boosting (XGBoost) machine learning model of the scikit-learning package applied to their eight previously and experimentally obtained PZQ cocrystals [ 69 72 ]. The cocrystal data in the CSD and the data recorded as no cocrystal formation in experimental screening were used as data sets for model training. The structures of the drug and the coformers were represented by simplified molecular input line entry specification (SMILES) strings. RDkit molecular descriptors computed from the input SMILES strings were used as the features of the corresponding compound, which were computed by the ChemDes website [ 128 ]. This model, applied to PZQ cocrystallization, predicted that PZQ could form cocrystals with all eight coformers, which was consistent with experimental results [ 69 72 ], thus revealing the model as a powerful tool for cocrystal prediction and design in the field of drug research.
Coming back to experimentally discovered PZQ cocrystals, D&#;Abbrunzo and coworkers recently reported a drug&#;drug antiparasitic cocrystal presenting a very peculiar stoichiometry, almost unusual in the variety of cocrystals known in the literature. Precisely, the novel cocrystal was obtained by grinding PZQ and Niclosamide (NCM) in a 1:3 molar ratio in the presence of a catalytic amount of MeOH for 120 min at 25 Hz [ 73 ].
1/c
, showing one PZQ and three NCM crystallographic independent molecules in the ASU (syn
conformation, contrary to most of PZQ multicomponent systems discussed in this review. Also, a low-temperature crystal structure was deposited in the CSD and indexed as RIPFOP.SEM images showed that the new cocrystal consisted of agglomerates of small plates whose particle size roughly varies in the range of 150&#; nm (see Figure 4 ). The DSC curve only showed an endothermic peak at 202.89 °C, attributable to the PZQ-NCM melting point and intermediate to those of pure PZQ and NCM (141.99 and 229.98 °C, respectively [ 59 129 ]). In addition, the laboratory diffraction pattern of the PZQ-NCM cocrystal showed reflections clearly different from those of the starting materials and in good agreement with the PXRD simulated from single crystals. The latter were obtained through conventional solution crystallization in EA starting from preformed seeds of PZQ-NCM cocrystal and analyzed through Synchrotron X-ray diffraction. The new cocrystal (indexed as RIPFOP01 in the CSD) crystallizes in the monoclinic unit cell with a space group of P2, showing one PZQ and three NCM crystallographic independent molecules in the ASU ( Figure 10 ). The centrosymmetric crystal packing is consistent with the presence of racemic PZQ, which is also partially disordered, in the unit cell. Structural analysis revealed that PZQ molecules exhibit a perpendicular relative orientation with respect to NCM molecules and all NCM molecules adopt a rigid, extended, and planar conformation defined by central amidic bond planarity constrain and homomolecular H-bonds in the crystal packing. The two carbonyl groups of PZQ act as H-acceptors bound to NCM donor hydroxyl groups and the third NCM present in the ASU is linked to the carbonyl of one NCM bound to PZQ. Interestingly, as in case of PZQ Form A, both PZQ carbonyl groups are oriented in theconformation, contrary to most of PZQ multicomponent systems discussed in this review. Also, a low-temperature crystal structure was deposited in the CSD and indexed as RIPFOP.
&#;1 and 897 cm&#;1, respectively [&#;1 was markedly shifted in the cocrystal, forming a doublet at and cm&#;1. Further, the signal of NCM C-OH stretching could be seen at cm&#;1, shifted at higher intensities in comparison to the raw peak at cm&#;1 [Intermolecular interactions and stoichiometry were also confirmed through FT-IR and SSNMR analyses. Considering the FT-IR results, the N-H stretching and bending peaks of NCM, originally at &#; cmand 897 cm, respectively [ 130 ], were downward shifted, suggesting the involvement of the NCM N-H group in H-bonds with PZQ. Moreover, the carbonyl stretching vibration of PZQ at cmwas markedly shifted in the cocrystal, forming a doublet at and cm. Further, the signal of NCM C-OH stretching could be seen at cm, shifted at higher intensities in comparison to the raw peak at cm 131 ].
Passing to the 13C CPMAS SSNMR spectrum, no traces of unreacted starting materials or PZQ polymorphs were detected, and a new crystalline phase was observed. Two isolated and split signals at about 153 and 175.6 ppm, respectively attributable to NCM and PZQ, were used to evaluate the stoichiometry ratio of the cocrystal: the integration of these two signals agreed with the presence of one PZQ and three NCM per ASU. In the 15N CPMAS spectrum, the N amide and nitro group chemical shifts demonstrated the formation of supramolecular interactions between PZQ and NCM and a new crystalline packing.
50,Also, the physical and chemical stability of PZQ-NCM were investigated under several conditions. As in previous works, a diminished PZQ recovery was noticed with the insurgence of peculiar degradation products as a function of the excipient used in binary ground systems [ 33 52 ], and the chemical stability of the cocrystal was examined through spectrometric evaluations: no typical PZQ tendency of decay was noticed in the experimental grinding conditions.
Physical stability was evaluated both at the solid state and in aqueous solution. The PZQ-NCM cocrystal remained unchanged over a period of 12 months at ambient temperature with no signs of dissociation into the parent compounds. In the context of physical stability in aqueous solution, thanks to the strong supramolecular interactions, the cocrystal structure confers a resistance to the otherwise predominant transition of NCM into the insoluble and undesired monohydrate NCM Ha, which usually arises within 1 month of NCM storage at ambient temperature [ 129 ].
Schistosoma mansoni
models compared to the corresponding physical mixture (PM). Based on these promising results, in vivo preliminary tests were also carried out: the new solid was administered as a powder in minicapsule size M (specific for mice) instead of the conventional aqueous suspensions commonly used during in vivo administration [More importantly, the PZQ-NCM cocrystal exhibited higher anthelmintic activity (%-effect of activity reduction) against in vitro adultmodels compared to the corresponding physical mixture (PM). Based on these promising results, in vivo preliminary tests were also carried out: the new solid was administered as a powder in minicapsule size M (specific for mice) instead of the conventional aqueous suspensions commonly used during in vivo administration [ 94 ]. Despite the limited number of mice treated, there was no significant difference between the number of worms recovered from infected mice treated with PZQ-NCM cocrystal and those with PM. In the case of treatment with pure PZQ, a comparison with the cocrystal was ineffective due to an underdosage compared to PZQ monotherapy. However, the administration of pure PZQ to mice resulted a positive control group, since it confirmed that the treatment with minicapsules worked, revealing cocrystal higher doses encouraging for future in vivo studies.
Pbca
unit cell, with the acidic oxygen of the carboxylic acid bridging between two different carbonyl oxygen atoms of two racemic PZQs. The hydroxyl group of MAL acts as an H-bond donor in an intramolecular H-bond for the adjacent carboxylic group, while it acts as an H-bond acceptor in an additional H-bond with another MAL from the nearest chains aligned in an antiparallel orientation in the crystal. PZQ carbonyl groups displayed the most commonanti
-conformation.A different approach to cocrystal screening protocols was reported by Cappuccino et al., who, instead of searching for other coformers for PZQ cocrystallization, investigated cocrystalline solid solution formation of PZQ in the presence of enantiomerically pure malic (MAL) and tartaric (TA) acids in scalemic and racemic stoichiometry [ 74 ]. Two new cocrystals were structurally characterized and deposited in the CSD and three non-stoichiometric mixed crystal forms were identified and isolated. Based on a previous work that reported the 1:1 cocrystallization of (RS)-PZQ in the presence of enantiomerically pure L-MAL, which allowed the resolution of two diastereomeric cocrystals (i.e., (R)-PZQ/L-MAL and (S)-PZQ/L-MAL) [ 68 ], in this work the authors investigated the crystallization of PZQ in the presence of racemic MA and obtained a four-component new cocrystal, (R)-PZQ/(S)-PZQ/D-MAL/L-MAL. Its crystal structure (CCDC deposition number ) was solved by PXRD, as no single crystals were grown. PZQ and MAL molecules alternate into racemic H-bonded chains along the b axis of the orthorhombicunit cell, with the acidic oxygen of the carboxylic acid bridging between two different carbonyl oxygen atoms of two racemic PZQs. The hydroxyl group of MAL acts as an H-bond donor in an intramolecular H-bond for the adjacent carboxylic group, while it acts as an H-bond acceptor in an additional H-bond with another MAL from the nearest chains aligned in an antiparallel orientation in the crystal. PZQ carbonyl groups displayed the most common-conformation.
A similar four-component phase was obtained by grinding PZQ in the presence of racemic TA (i.e., (R)-PZQ/(S)-PZQ/D-TA/L-TA). In this crystal structure (CCDC deposition number ), chains of (S)-PZQ and L-TA extend along an axis of the triclinic P-1 unit cell and alternate with homologous chains of (R)-PZQ and D-TA kept together by the same monodentate interaction between the TA acidic group and the PZQ carbonyl group. Adjacent homochiral chains are held together by intermolecular H-bonds involving hydroxyl groups. Even in this case, PZQ molecules presented an
anti
-conformation.In case of using TA, no structure was determined from the mechanochemical crystallization of (RS)-PZQ in the presence of D-TA, so it remained unclear whether the system was a three-component system (i.e., (R)-PZQ/(S)-PZQ/D-TA) or a mixture of diastereomeric crystals (i.e., (R)-PZQ/D-TA and (S)-PZQ/D-TA), as for MAL.
Subsequently, the authors tried scalemic mixtures and substitutions between MAL and TA to assess the formation of (RS)-PZQ solid solutions with varied acid compositions.
Precisely, milling (RS)-PZQ in the presence of scalemic ratios of MAL (i.e., (R)-PZQ/(S)-PZQ/(D)-MAL/(L)-MAL = 1.5:1.5:1:2) did not give (R)-PZQ/L-MAL and (S)-PZQ/L-MAL, whereas it formed (R)-PZQ/(S)-PZQ/D-MAL/L-MAL, pointing toward the formation of a solid solution. DSC curves showed, indeed, a superimposition between the peak observed for (R)-PZQ/(S)-PZQ/D-MAL/L-MAL and the obtained solid solution.
A similar result was observed in the case of TA: grinding (RS)-PZQ in the presence of D-TA and L-TA in a 3:1:2 ratio (i.e., (R)-PZQ/(S)-PZQ/(D)-TA/(L)-TA = 1.5:1.5:1:2) gave (R)-PZQ/(S)-PZQ/D-TA/L-TA, as also confirmed by DSC results.
Moreover, the LAG of (RS)-PZQ with homochiral D-TA and L-MAL in a 1.5:1.5:1:2 ratio did not provide a solid solution but gave a mixture of (R)-PZQ/L-MAL and (S)-PZQ/L-MAL and the above-mentioned unknown product with D-TA.
When (RS)-PZQ was milled in the presence of the pseudoracemate (1:1 mixture) of L-MAL and L-TA, the unknown product with D-TA was obtained and the same was observed even by increasing the amount of L-TA compared to that of L-MAL, suggesting that a four-component cocrystalline solid solution is obtained at a high TA content. (R)-PZQ/L-MAL and (S)-PZQ/L-MAL only appeared at the higher ratio of L-MAL.
The authors also highlighted the possibility of obtaining a five-component solid solution by substituting part of L-MAL with D-MAL in the unknown product with D-TA (to obtain (R)-PZQ/(S)-PZQ/D-MAL/L-MAL/L-TA) or part of D-TA with L-MAL in (R)-PZQ/(S)-PZQ/D-TA/L-TA (to obtain (R)-PZQ/(S)-PZQ/L-MAL/D-TA/L-TA). Instead, physical mixtures were obtained while attempting the substitution of D-MAL with L-TA in the cocrystal (R)-PZQ/(S)-PZQ/D-MAL/L-MAL (to obtain (R)-PZQ/(S)-PZQ/D-MAL/L-MAL/L-TA) and the solid solution with the six components together.
Furthermore, solubility analysis indicated a four-fold solubility advantage for the newly prepared solid solutions over the pure drug and a faster dissolution rate, probably due to the metastable character of these solid solutions. To evaluate whether the increase in solubility and dissolution rate translated into higher oral bioavailability, in vivo pilot tests were performed on rats by administering solid solutions as powder for the first time in minicapsule size nine. Solid solutions demonstrated a faster absorption compared to the pure drug and helped to maintain a constant steady-state concentration.
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