Search

Anna Stasiłowicz,1 Ewa Tykarska,2 Natalia Rosiak,1 Kinga Sałat,3 Anna Furgała-Wojas,3 Tomasz Plech,4 Kornelia Lewandowska,5 Katarzyna Pikosz,1 Kamil Pawłowicz,1 Judyta Cielecka-Piontek1 1Department of Pharmacognosy, Poznan University of Medical Sciences, Poznan, Poland; 2Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Poznan, Poland; 3Department of Pharmacodynamics, Jagiellonian University Medical College, Krakow, Poland; 4Department of Pharmacology, Medical University of Lublin, Lublin, Poland; 5Institute of Molecular Physics, Polish Academy of Sciences, Poznan, PolandCorrespondence: Judyta Cielecka-PiontekDepartment of Pharmacognosy, Poznan University of Medical Sciences, Swiecickiego 4, 61– 781, Poznan, PolandTel +48 61 854 67 09Fax +48 61 854 67 01Email jpiontek@ump.edu.plPurpose: The poorly soluble nonsteroidal anti-inflammatory drug (NSAID), tolfenamic acid (TA), was studied to maximize its solubility, permeability through biological membranes, and pharmacological activity.Methods: A mixture with magnesium stearate (MS) – microenvironment pH-modifier was prepared, as well as systems additionally containing incorporating substances methyl-β-cyclodextrin (M-β-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). The identification of TA-MS-CD systems was confirmed using experimental methods: X-ray powder diffraction (XRPD) and Fourier transform infrared spectroscopy (FT-IR) with the theoretical support. Apparent solubility study was performed using the paddle apparatus, while in vitro gastrointestinal tract (GIT) and blood-brain barrier (BBB) permeability were conducted by using PAMPA (Parallel Artificial Membrane Permeability Assay). The in vivo part of the study used the mouse nitroglycerin (NTG)-induced migraine pain model.Results: From practically insoluble substance, TA in TA-MS-M-β-CD system dissolved up to 80.13% ± 2.77%, and in TA-MS-HP-β-CD up to 92.39% ± 3.25% in 180 minutes. An increase in TA permeability was also obtained in the TA-MS-M-β-CD and TA-MS-HP-β-CD systems through GIT membranes (Papp values 2.057 x 10− 5 cm s− 1 and 2.091 x 10− 5 cm s− 1, respectively) and through BBB (Papp values 3.658 x 10− 5 cm s− 1 and 3.629 x 10− 5 cm s− 1, respectively). The enlargement of the solubility and permeability impacted analgesia. The dose 25 mg/kg of both TA-MS-HP-β-CD and TA-MS-M-β-CD was almost equally effective and only slightly less effective than the dose 50 mg/kg of pure TA. Both TA-MS-HP-β-CD and TA-MS-M-β-CD used at 50 mg/kg more effectively attenuated tactile allodynia in NTG-treated mice than the same dose of pure TA. None of TA forms influenced heat hyperalgesia.Conclusion: Increasing solubility of TA caused an increase of its analgesic effect in an animal model of migraine pain.Keywords: tolfenamic acid, migraine pain, cyclodextrins, permeability, gastrointestinal tract, blood-brain barrier

Apigenin (APG), a bioactive flavonoid with promising therapeutic potential, suffers from poor water solubility, which limits its bioavailability. To address this, solid dispersions of APG were prepared using ball milling with sodium alginate (SA), Pluronic ® F-68 (PLU68), Pluronic ® F-127 (PLU127), PVP K30, and PVP VA64 as polymeric excipients. These dispersions were screened for apparent solubility in water and buffers with pH 1.2, 5.5, and 6.8. Based on improved solubility after 60 min, APG-PLU68 and APG-PLU127 dispersions were selected for further study. DSC and FT-IR analysis confirmed molecular interactions between APG and the polymer matrices, contributing to enhanced solubility and dissolution rates. Dissolution rate studies showed that APG-PLU127 achieved 100% solubility at pH 6.8, suggesting its potential use in environments such as the small intestine. Additionally, APG-PLU127 exhibited 84.3% solubility at pH 1.2, indicating potential for solid oral dosage forms, where APG could be absorbed in the acidic conditions of the stomach. The stability study confirmed that storage for one year under ambient conditions does not cause chemical degradation but affects the physical state and solubility of the dispersion. Antioxidant activity was assessed using the ABTS assay. Freshly obtained APG-PLU127 showed 68.1% ± 1.94% activity, whereas APG-PLU127 stored for one year under ambient conditions exhibited 66.2% ± 1.62% (significant difference, p < 0.05). The difference was related to a slight decrease in the solubility of APG in the solid dispersion (T0 = 252 ± 1 μg∙mL -1 , T1 = 246 ± 1 μg∙mL -1 ). The findings demonstrate the superior performance of PLU127 as a carrier for enhancing the solubility, release, and antioxidant activity of APG.

In this study, binary amorphous solid dispersions (ASDs, fisetin-Eudragit ® ) and ternary amorphous solid inclusions (ASIs, fisetin-Eudragit ® -HP-β-cyclodextrin) of fisetin (FIS) were prepared by the mechanochemical method without solvent. The amorphous nature of FIS in ASDs and ASIs was confirmed using XRPD (X-ray powder diffraction). DSC (Differential scanning calorimetry) confirmed full miscibility of multicomponent delivery systems. FT-IR (Fourier-transform infrared analysis) confirmed interactions that stabilize FIS's amorphous state and identified the functional groups involved. The study culminated in evaluating the impact of amorphization on water solubility and conducting in vitro antioxidant assays: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-ABTS, 2,2-diphenyl-1-picrylhydrazyl-DPPH, Cupric Reducing Antioxidant Capacity-CUPRAC, and Ferric Reducing Antioxidant Power-FRAP and in vitro neuroprotective assays: inhibition of acetylcholinesterase-AChE and butyrylcholinesterase-BChE. In addition, molecular docking allowed for the determination of possible bonds and interactions between FIS and the mentioned above enzymes. The best preparation turned out to be ASI&#95;30&#95;EPO (ASD fisetin-Eudragit ® containing 30% FIS in combination with HP-β-cyclodextrin), which showed an improvement in apparent solubility (126.5 ± 0.1 µg∙mL -1 ) and antioxidant properties (ABTS: IC 50 = 10.25 µg∙mL -1 , DPPH: IC 50 = 27.69 µg∙mL -1 , CUPRAC: IC 0.5 = 9.52 µg∙mL -1 , FRAP: IC 0.5 = 8.56 µg∙mL -1 ) and neuroprotective properties (inhibition AChE: 39.91%, and BChE: 42.62%).

Our research aimed to develop an amorphous solid dispersion (ASD) of myricetin (MYR) with Polyvinylpyrrolidone K30 (PVP30) to enhance its solubility, dissolution rate, antioxidant, and neuroprotective properties. Employing a combination of solvent evaporation and freeze drying, we successfully formed MYR ASDs. XRPD analysis confirmed complete amorphization in 1:8 and 1:9 MYR-PVP weight ratios. DSC thermograms exhibited a single glass transition (Tg), indicating full miscibility. FT-IR results and molecular modeling confirmed hydrogen bonds stabilizing MYR's amorphous state. HPLC analysis indicated the absence of degradation products, ensuring safe MYR delivery systems. Solubility, dissolution rate (pH 1.2 and 6.8), antioxidant (ABTS, DPPH, CUPRAC, and FRAP assays), and in vitro neuroprotective activities (inhibition of cholinesterases: AChE and BChE) were significantly improved compared to the pure compound. Molecular docking studies revealed that MYR had made several hydrogen, hydrophobic, and π-π stacking interactions, which could explain the compound's potency to inhibit AChE and BChE. MYR-PVP 1:9 w / w ASD has the best solubility, antioxidant, and neuroprotective activity. Stability studies confirmed the physical stability of MYR-PVP 1:9 w / w ASD immediately after dissolution and for two months under ambient conditions. Our study showed that the obtained ASDs are promising systems for the delivery of MYR with the potential for use in alleviating the symptoms of neurodegenerative diseases.

In this study, amorphous solid dispersions (ASDs) of pterostilbene (PTR) with polyvinylpyrrolidone polymers (PVP K30 and VA64) were prepared through milling, affirming the amorphous dispersion of PTR via X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Subsequent analysis of DSC thermograms, augmented using mathematical equations such as the Gordon-Taylor and Couchman-Karasz equations, facilitated the determination of predicted values for glass transition (T g ), PTR's miscibility with PVP, and the strength of PTR's interaction with the polymers. Fourier-transform infrared (FTIR) analysis validated interactions maintaining PTR's amorphous state and identified involved functional groups, namely, the 4'-OH and/or -CH groups of PTR and the C=O group of PVP. The study culminated in evaluating the impact of amorphization on water solubility, the release profile in pH 6.8, and in vitro permeability (PAMPA-GIT and BBB methods). In addition, it was determined how improving water solubility affects the increase in antioxidant (ABTS, DPPH, CUPRAC, and FRAP assays) and neuroprotective (inhibition of cholinesterases: AChE and BChE) properties. The apparent solubility of the pure PTR was ~4.0 µg·mL -1 and showed no activity in the considered assays. For obtained ASDs (PTR-PVP30/PTR-PVPVA64, respectively) improvements in apparent solubility (410.8 and 383.2 µg·mL -1 ), release profile, permeability, antioxidant properties (ABTS: IC 50 = 52.37/52.99 μg·mL -1 , DPPH: IC 50 = 163.43/173.96 μg·mL -1 , CUPRAC: IC 0.5 = 122.27/129.59 μg·mL -1 , FRAP: IC 0.5 = 95.69/98.57 μg·mL -1 ), and neuroprotective effects (AChE: 39.1%/36.2%, BChE: 76.9%/73.2%) were confirmed.

Attenuated total reflection-Mid-Fourier transform-infrared (ATR-Mid-FT-IR) spectroscopy combined with principal component analysis (PCA) has been applied for the discrimination of amorphous solid dispersion (ASD) of kaempferol with different types of Eudragit (L100, L100-55, EPO). The ASD samples were prepared by ball milling. Training and test sets for PCA consisted of a pure compound, physical mixture, and incomplete/complete amorphous solid dispersion. The obtained results confirmed that the range 400-1700 cm -1 was the major contributor to the variance described by PC1 and PC2, which are the fingerprint region. The obtained PCA model selected fully amorphous samples as follows: five for KMP-EL100, two for KMP-EL100-55, and six for KMP-EPO (which was confirmed by the XRPD analysis). DSC analysis confirmed full miscibility of all ASDs (one glass transition temperature). FT-IR analysis confirmed the formation of hydrogen bonds between the -OH and/or -CH groups of KMP and the C=O group of Eudragits. Amorphization improved the solubility of kaempferol in pH 6.8, pH 5.5, and HCl 0.1 N.

Poor bioavailability hampers the use of curcumin and piperine as biologically active agents. It can be improved by enhancing the solubility as well as by using bioenhancers to inhibit metabolic transformation processes. Obtaining an amorphous system of curcumin and piperine can lead to the overcoming of these limitations. Hot-melt extrusion successfully produced their amorphous systems, as shown by XRPD and DSC analyses. Additionally, the presence of intermolecular interactions between the components of the systems was investigated using the FT-IR/ATR technique. The systems were able to produce a supersaturation state as well as improve the apparent solubilities of curcumin and piperine by 9496- and 161-fold, respectively. The permeabilities of curcumin in the GIT and BBB PAMPA models increased by 12578- and 3069-fold, respectively, whereas piperine's were raised by 343- and 164-fold, respectively. Improved solubility had a positive effect on both antioxidant and anti-butyrylcholinesterase activities. The best system suppressed 96.97 ± 1.32% of DPPH radicals, and butyrylcholinesterase activity was inhibited by 98.52 ± 0.87%. In conclusion, amorphization remarkably increased the dissolution rate, apparent solubility, permeability, and biological activities of curcumin and piperine.

The aim of our research was to improve the solubility and antioxidant activity of pterostilbene (PTR) by developing a novel amorphous solid dispersion (ASD) with Soluplus ® (SOL). DSC analysis and mathematical models were used to select the three appropriate PTR and SOL weight ratios. The amorphization process was carried out by a low-cost and green approach involving dry milling. An XRPD analysis confirmed the full amorphization of systems in 1:2 and 1:5 weight ratios. One glass transition (T g ) observed in DSC thermograms confirmed the complete miscibility of the systems. The mathematical models indicated strong heteronuclear interactions. SEM micrographs suggest dispersed PTR within the SOL matrix and a lack of PTR crystallinity, and showed that after the amorphization process, PTR-SOL systems had a smaller particle size and larger surface area compared with PTR and SOL. An FT-IR analysis confirmed that hydrogen bonds were responsible for stabilizing the amorphous dispersion. HPLC studies showed no decomposition of PTR after the milling process. PTR's apparent solubility and antioxidant activity after introduction into ASD increased compared to the pure compound. The amorphization process improved the apparent solubility by ~37-fold and ~28-fold for PTR-SOL, 1:2 and 1:5 w / w , respectively. The PTR-SOL 1:2 w / w system was preferred due to it having the best solubility and antioxidant activity (ABTS: IC 50 of 56.389 ± 0.151 µg·mL -1 and CUPRAC: IC 0.5 of 82.52 ± 0.88 µg·mL -1 ).

Oleanolic (OA) and glycyrrhetinic acids (GE), as well as their derivatives, show a variety of pharmacological properties. Their crystal structures provide valuable information related to the assembly modes of these biologically active compounds. In the known-to-date crystals of OA esters, their 11-oxo derivatives, and GE ester crystals, triterpenes associate, forming different types of ribbons and layers whose construction is based mainly on van der Waals forces and weak C-H···O interactions. New crystal structures of 11-oxo OA methyl ester and the polymorph of OA butyl ester reveal an alternative aggregation mode. Supramolecular architectures consist of helical chains which are stabilized by hydrogen bonds of O-H···O type. It was found that two polymorphic forms of butyl OA ester (layered and helical) are related monotropically. In a structure of metastable form, O-H···O hydrogen bonds occur, while the thermodynamically preferred phase is governed mainly by van der Waals interactions. The intermolecular interaction energies calculated using CrystalExplorer, PIXEL, and Psi4 programs showed that even in motifs formed through O-H···O hydrogen bonds, the dispersive forces have a significant impact.

The objective of this study was to obtain co-amorphous systems of poorly soluble sinapic acid using amino acids as co-formers. In order to assess the probability of the interaction of amino acids, namely, arginine, histidine, lysine, tryptophan, and proline, selected as co-formers in the amorphization of sinapic acid, in silico studies were carried out. Sinapic acid systems with amino acids in a molar ratio of 1:1 and 1:2 were obtained using ball milling, solvent evaporation, and freeze drying techniques. X-ray powder diffraction results confirmed the loss of crystallinity of sinapic acid and lysine, regardless of the amorphization technique used, while remaining co-formers produced mixed results. Fourier-transform infrared spectroscopy analyses revealed that the co-amorphous sinapic acid systems were stabilized through the creation of intermolecular interactions, particularly hydrogen bonds, and the potential formation of salt. Lysine was selected as the most appropriate co-former to obtain co-amorphous systems of sinapic acid, which inhibited the recrystallization of sinapic acid for a period of six weeks in 30 °C and 50 °C. Obtained co-amorphous systems demonstrated an enhancement in dissolution rate over pure sinapic acid. A solubility study revealed a 12.9-fold improvement in sinapic acid solubility after introducing it into the co-amorphous systems. Moreover, a 2.2-fold and 1.3-fold improvement in antioxidant activity of sinapic acid was observed with respect to the ability to neutralize the 2,2-diphenyl-1-picrylhydrazyl radical and to reduce copper ions, respectively.

Despite the recent dynamic development of medicine, influenza is still a significant epidemiological problem for people around the world. The growing resistance of influenza viruses to currently available antiviral drugs makes it necessary to search for new compounds or drug forms with potential high efficacy against human influenza A and B viruses. One of the methods of obtaining new active compounds is to chemically modify privileged structures occurring in the natural environment. The second solution, that is gaining more and more interest, is the use of modern drug carriers, which significantly improve physicochemical and pharmacokinetic parameters of the transported substances. Molecules known from the earliest times for their numerous therapeutic properties are glycyrrhizinic acid (GA) and glycyrrhetinic acid (GE). Both compounds constitute the main active agents of the licorice (Glycyrrhiza glabra, Leguminosae) root and, according to a number of scientific reports, show antiviral properties against both DNA and RNA viruses. The above information prompted many scientific teams around the world to obtain and test in vitro and/or in vivo new synthetic GA and GE derivatives against influenza A and B viruses. Similarly, in recent years, a significant amount of GA and GE-based drug delivery systems (DDS) such as nanoparticles, micelles, liposomes, nanocrystals, and carbon dots has been prepared and tested for antiviral activity, including those against influenza A and B viruses. This work systematizes the attempts undertaken to study the antiviral activity of new GA and GE analogs and modern DDS against clinically significant human influenza viruses, at the same time indicating the directions of their further development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

The present study reports amorphous solid dispersions (ASDs) of hesperidin (Hes) prepared by ball milling to improve its solubility and apparent solubility over the unmodified compound. The carriers were Soluplus ® (Sol), alginate sodium (SA), and hydroxypropylmethylcellulose (HPMC). XRPD analysis confirmed full amorphization of all binary systems in 1:5 w/w ratio. One glass transition (T g ) observed in DSC thermograms of hesperidin:Soluplus ® (Hes:Sol) and hesperidin:HPMC (Hes:HPMC) 1:5 w/w systems confirmed complete miscibility. The mathematical model (Gordon-Taylor equation) indicates that the obtained amorphous systems are characterized by weak interactions. The FT-IR results confirmed that hydrogen bonds are responsible for stabilizing the amorphous state of Hes. Stability studies indicate that the strength of these bonds is insufficient to maintain the amorphous state of Hes under stress conditions (25 °C and 60 °C 76.4% RH). HPLC analysis suggested that the absence of degradation products indicates safe hesperidin delivery systems. The solubility and apparent solubility were increased in all media (water, phosphate buffer pH 6.8 and HCl (0.1 N)) compared to the pure compound. Our study showed that all obtained ASDs are promising systems for Hes delivery, wherein Hes:Sol 1:5 w/w has the best solubility (about 300-fold in each media) and apparent solubility (about 70% in phosphate buffer pH 6.8 and 63% in HCl).

Iron(III) porphyrazines containing peripheral 2,5-dimethyl-, 2-methyl-5-phenyl-, and 2,3,5-triphenyl-1 H -pyrrol-1-yl substituents were synthesized and subjected to physicochemical characterization. This was accomplished by high-resolution mass spectrometry, nuclear magnetic resonance (as diamagnetic Fe(II) derivatives), HPLC purity analysis, and UV-Vis spectroscopy, accompanied by the solvation study in dichloromethane and pyridine. X-ray structure analysis was performed for a single crystal of the previously obtained 2,5-diphenyl-substituted derivative of porphyrazine complex ( 5d ). The octahedral geometries of iron cation, present in the porphyrazine core, influenced the packing mode of molecules in the crystals. Mössbauer studies, performed for solid samples of iron porphyrazines, indicated that low-spin reduced iron states might occupy low- or high-symmetry binding sites. It was found that the hyperfine parameters and the subsequent contribution of the iron cations depend on the number of phenyl groups surrounding the pyrrolyl moiety. For iron(II) porphyrazine 2,3,5-triphenylpyrrol-1-yl substituents ( 5b ), a high-spin ferrous state fraction was observed. Temperature-dependent measurements showed that the freed rotation of the peripheral porphyrazine ligands and the increased flexibility of the macrocycle ring result in the Fe 2+ ion being stabilized in a diamagnetic state at a binding site of high symmetry at room temperature in the solid state. This process is most probably stimulated by the range of collective motions of the polymeric ribbons consisting of iron(II) porphyrazines observed in the X-ray.

Magnesium(II) sulfanyl porphyrazine with peripheral morpholinethoxy substituents was embedded on the surface of titanium(IV) dioxide nanoparticles. The obtained nanocomposites were characterized with the use of particle size and distribution (NTA analysis), electron microscopy (SEM), thermal analysis (TGA), FTIR-ATR spectroscopy, and X-ray powder diffraction (XRD). The measured particle size of the obtained material was 327.4 ± 15.5 nm. Analysis with XRD showed no visible changes in the crystallinity of the material after deposition of porphyrazine on the TiO 2 surface. However, SEM images revealed noticeable changes in the morphology of the obtained hybrid material: higher aggregation and less ordered structure of the aggregates. The TGA analysis revealed the lost 3.6% (0.4 mg) of the mass of obtained material in the range 250-550 °C. In the FTIR-ATR analysis, C-H stretching vibratins in the range of 3000-2800 cm -1 , originating from porphyrazine moieties, were detected. The photocatalytic applicability of the nanomaterial was assessed in photodegradation studies of methylene blue and bisphenol A as reference environmental pollutants. In addition, the photocatalytic degradation of carbamazepine with porphyrazine/TiO 2 hybrids as photocatalysts was studied, accompanied by an HPLC chromatography assessment of photodegradation. In total, 43% of the initial concentration was achieved in the case of bisphenol A, after 4 h of irradiation, whereas 57% was achieved in the case of carbamazepine. In each photodegradation reaction, the activity of the obtained photocatalytic nanomaterial was proved with almost linear degradation. The photodegradation reaction rate constants were calculated, and revealed 5.75 × 10 -5 s -1 for bisphenol A and 5.66 × 10 -5 s -1 for carbamazepine.

Rosmarinic acid (RA) is a natural antioxidant with neuroprotective properties; however, its preventive and therapeutic use is limited due to its slight solubility and poor permeability. This study aimed to improve RA physicochemical properties by systems formation with cyclodextrins (CDs): hydroxypropyl-α-CD (HP-α-CD), HP-β-CD, and HP-γ-CD, which were prepared by the solvent evaporation (s.e.) method. The interactions between components were determined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC) and Fourier Transform infrared spectroscopy (FTIR). The sites of interaction between RA and CDs were suggested as a result of in silico studies focused on assessing the interaction between molecules. The impact of amorphous systems formation on water solubility, dissolution rate, gastrointestinal (GIT) permeability, and biological activity was studied. RA solubility was increased from 5.869 mg/mL to 113.027 mg/mL, 179.840 mg/mL, and 194.354 mg/mL by systems formation with HP-α-CD, HP-β-CD, and HP-γ-CD, respectively. During apparent solubility studies, the systems provided an acceleration of RA dissolution. Poor RA GIT permeability at pH 4.5 and 5.8, determined by parallel artificial membrane permeability assay (PAMPA system), was increased; RA-HP-γ-CD s.e. indicated the greatest improvement (at pH 4.5 from P app 6.901 × 10 -7 cm/s to 1.085 × 10 -6 cm/s and at pH 5.8 from 5.019 × 10 -7 cm/s to 9.680 × 10 -7 cm/s). Antioxidant activity, which was determined by DPPH, ABTS, CUPRAC, and FRAP methods, was ameliorated by systems; the greatest results were obtained for RA-HP-γ-CD s.e. The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was increased from 36.876% for AChE and 13.68% for BChE to a maximum inhibition of the enzyme (plateau), and enabled reaching IC 50 values for both enzymes by all systems. CDs are efficient excipients for improving RA physicochemical and biological properties. HP-γ-CD was the greatest one with potential for future food or dietary supplement applications.

Synthesis and structural characterization of new esters of oleanolic acid and its 11-oxo derivatives are reported. Compounds crystallize in four isostructural groups, each containing one to four structures. Single-crystal X-ray analysis revealed that molecules belonging to non-isostructural groups self-associate according to two schemes that describe also supramolecular architectures in crystals of glycyrrhetinic acid derivatives. Structural motifs arise as a result of van der Waals forces. Parameters introduced for the analysis of one- and two-dimensional assemblies allow the comparison of motifs in isostructural and non-isostructural crystals, including polymorphs, and a qualitative assessment of differences in molecular self-assembly. One-, two- or three-dimensional similarity has been confirmed by XPac calculations.

Due to the destruction of the integrity of the parent crystal, single-crystal-to-single-crystal phase transition in organic compounds is still a relatively rare phenomenon. The phase transition in glycyrrhetinic acid isopropyl ester is triggered by temperature change. The increasing volume of the isopropyl substituent as a result of increasing temperature forces a remodelling of the structural motifs. These changes cause a single-crystal-to-single-crystal phase transition. The low-temperature form is isostructural with glycyrrhetinic acid methanol solvate, while the high-temperature phase is isostructural with the ethyl ester of this acid.

Newly developed tetra- and octasubstituted methimazole-phthalocyanine conjugates as potential photosensitizers have been obtained. Synthesized intermediates and final products were characterized by the MALD-TOF technique and various NMR techniques, including 2D methods. Single-crystal X-ray diffraction was used to determine the crystal structures of dinitriles. The studied phthalocyanines revealed two typical absorption bands-the Soret band and the Q band. The most intense fluorescence was observed for octasubstituted magnesium(II) phthalocyanine in DMF (Φ FL = 0.022). The best singlet oxygen generators were octasubstituted magnesium(II) and zinc(II) phthalocyanines (Φ ∆ 0.56 and 0.81, respectively). The studied compounds presented quantum yields of photodegradation at the level between 10 -5 and 10 -6 . Due to their low solubility in a water environment, the liposomal formulations were prepared. Within the studied group, octasubstituted zinc(II) phthalocyanine at the concentration of 100 µM activated with red light showed the highest antibacterial activity against S. aureus equal to a 5.68 log reduction of bacterial growth.

This study aimed at obtaining hesperidin (Hed) and hesperetin (Het) systems with HP-β-CD by means of the solvent evaporation method. The produced systems were identified using infrared spectroscopy (FT-IR), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC). Moreover, in silico docking and molecular dynamics studies were performed to assess the most preferable site of interactions between tested compounds and HP-β-CD. The changes of physicochemical properties (solubility, dissolution rate, and permeability) were determined chromatographically. The impact of modification on biological activity was tested in an antioxidant study as well as with regards to inhibition of enzymes important in pathogenesis of neurodegenerative diseases. The results indicated improvement in solubility over 1000 and 2000 times for Hed and Het, respectively. Permeability studies revealed that Hed has difficulties in crossing biological membranes, in contrast with Het, which can be considered to be well absorbed. The improved physicochemical properties influenced the biological activity in a positive manner by the increase in inhibitory activity on the DPPH radical and cholinoesterases. To conclude the use of HP-β-CD as a carrier in the formation of an amorphous inclusion complex seems to be a promising approach to improve the biological activity and bioavailability of Hed and Het.

Piperine is an alkaloid that has extensive pharmacological activity and impacts other active substances bioavailability due to inhibition of CYP450 enzymes, stimulation of amino acid transporters and P -glycoprotein inhibition. Low solubility and the associated low bioavailability of piperine limit its potential. The combination of piperine with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) causes a significant increase in its solubility and, consequently, an increase in permeability through gastrointestinal tract membranes and the blood-brain barrier. X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) were used to characterize interactions between piperine and HP-β-CD. The observed physicochemical changes should be combined with the process of piperine and CD system formation. Importantly, with an increase in solubility and permeability of piperine as a result of interaction with CD, it was proven to maintain its biological activity concerning the antioxidant potential (2,2-diphenyl-1-picryl-hydrazyl-hydrate assay), inhibition of enzymes essential for the inflammatory process and for neurodegenerative changes (hyaluronidase, acetylcholinesterase, butyrylcholinesterase).

The health benefits of resveratrol have been proven to inhibit the development of numerous diseases. A frequent limitation in its use is a low bioavailability stemming from a poor solubility and fast enterohepatic metabolism. Thus, the aim of the research was to investigate the possibility to formulate mucoadhesive cyclodextrin- and xanthan gum-based buccal tablets in order to increase the solubility of resveratrol and to eliminate bypass enterohepatic metabolism. Systems of resveratrol with α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), γ-cyclodextrin (γ-CD) and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) prepared by the dry mixing method (ratio 1:1) were selected for the of tablets where xanthan gum was used as a mucoadhesive agent. They were identified on the basis of PXRD, FT-IR analysis. Tablets F1 (with α-CD), F2 (with β-CD) and F3 (with γ-CD) were characterized by the highest compactibility as well as by favorable mucoadhesive properties. Resveratrol release from these tablets was delayed and controlled by diffusion. The tablets prepared in the course of this study appear to constitute promising resveratrol delivery systems and are recommended to increase the effectiveness of the treatment in many diseases, particularly periodontitis.

Progressive increase in bacterial resistance has caused an urgent need to introduce new antibiotics, one of them being oxazolidinones with their representative tedizolid. Despite the broad spectrum of activity of the parent tedizolid, it is characterized by low water solubility, which limits its use. The combination of the active molecule with a multifunctional excipient, which is cyclodextrins, allows preservation of its pharmacological activity and modification of its physicochemical properties. Therefore, the aim of the study was to change the dissolution rate and permeability through the model membrane of tedizolid by formation of solid dispersions with a cyclodextrin. The research included identification of tedizolid-hydroxypropyl-β-cyclodextrin (tedizolid/HP-β-CD) inclusion complex by thermal method (Differential Scanning Colorimetry), spectroscopic methods (powder X-ray diffraction, Fourier-Transform Infrared spectroscopy), and molecular docking. The second part of the research concerned the physicochemical properties (dissolution and permeability) and the biological properties of the system in terms of its microbiological activity. An increase in the dissolution rate was observed in the presence of cyclodextrin, while maintaining a high permeation coefficient and high microbiological activity. The proposed approach is an opportunity to develop drug delivery systems used in the treatment of resistant bacterial infections, in which, in addition to modifying the physicochemical properties caused by cyclodextrin, we observe a favorable change in the pharmacological potential of the bioactives.

The nutraceutical system of curcumin-piperine in 2-hydroxypropyl-β-cyclodextrin was prepared by using the kneading technique. Interactions between the components of the system were defined by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR). Application of hydroxypropyl-β-cyclodextrin as a carrier-solubiliser improved solubility of the curcumin-piperine system, its permeability through biological membranes (gastrointestinal tract, blood-brain barrier) as well as the antioxidant, antimicrobial and enzyme inhibitory activities against acetylcholinesterase and butyrylcholinesterase.

This study aimed to investigate changes in the solubility and antimicrobial efficacy of cefuroxime axetil (CA) when incorporated into cyclodextrin (CD). While choosing the CD, the validated in silico model was used. A theoretical model based on docking and molecular mechanics/generalized born surface area was validated using a curated dataset of API (active pharmaceutical ingredient)-CD stability constants. The library of commonly used cyclodextrins was virtually screened, indicating CA -hydroxypropyl-βCD (HPβCD) as the most thermodynamically favored system. Solid-state CA-HPβCD system was prepared and characterized by differential scanning calorimetry (DSC), Fourier-transform infrared (FT-IR), and X-ray diffraction (XRPD) methods. The dissolution profiles of the CA and its cyclodextrin system were evaluated. Microbiological activity of the CA-HPβCD inclusion system was studied based on changes in minimal inhibitory concentration (MIC) values and related to ones of the pure CA. The theoretical model was successfully validated, obtaining an average correlation with experimental data R = 0.7. The dissolution study showed significantly improved dissolution profiles of CA-HPβCD compared to CA. HPβCD increases the antimicrobial efficacy of CA up to 4-fold compared to pure CA., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

For the first time, the influence of ionising radiation on the physicochemical properties of ertapenem in solid state was studied. During our studies, we evaluated the possibility of applying radiosterilization to obtain sterile ertapenem. Spectroscopic (Fourier Transform Infrared (FT-IR)), thermal (differential scanning calorimetry (DSC), chromatography (High-Performance Liquid Chromatography (HPLC) and HPLC-MS), and X-ray powder diffraction (XRPD) studies shown that irradiation of ertapenem with the 25 kGy, the dose required to achieve sterility, does not change the physicochemical properties of the studied compound. The antimicrobial activity of ertapenem irradiated with the dose of 25 kGy was only reduced for one species. Based on the received results, we can conclude that radiostelization is a promising alternative method of obtaining sterile ertapenem. In our studies, ertapenem was also exposed to e-beam radiation with a dose of 400 kGy. It was determined that two novel degradation products that are structurally differently to degradants formed during hydrolysis and thermolysis.

Photophysical properties and photodynamic antibacterial potential of magnesium phthalocyanines bearing 2-propoxy, benzyloxy, 3,5-bis(benzyloxy)benzyloxy substituents at non-peripheral positions were studied. The UV-Vis absorption spectra of researched phthalocyanine derivatives were found typical. Extension of peripheral substituent size from 2-propoxy to benzyloxy and finally 3,5-bis(benzyloxy)benzyloxy was accompanied by the rise of quantum yield of fluorescence up to 0.17 and 0.04 in DMF and DMSO, respectively. Similarly, the expansion of the phthalocyanine periphery from the 2-propoxy to benzyloxy and 3,5-bis(benzyloxy)benzyloxy groups resulted in a detectable increase of the singlet oxygen quantum yield values to 0.04, 0.12, 0.14 respectively, which was assessed following direct method of singlet oxygen phosphorescence measurement at 1270 nm. Studied phthalocyanines undergo photobleaching process with the quantum yields at the level of 10 -6 in DMSO and 10 -5 in DMF. The size of phthalocyanine impacted the process of liposomal formulation. Small liposome vesicles containing non-peripherally substituted phthalocyanines with 2-propoxy and benzyloxy substituents were obtained following extrusion method. The unification process of the liposomes loaded with 3,5-bis(benzyloxy)benzyloxy non-peripherally substituted phthalocyanines was not possible. In in vitro antimicrobial photodynamic inactivation study, log reduction values of bacterial (Enterococcus faecalis) growth at 3.61 and 2.99 were achieved for liposomal formulations containing phthalocyanines with 2-propoxy and benzyloxy substituents respectively, whereas phthalocyanine with 3,5-bis(benzyloxy)benzyloxy substituents was inactive. Phthalocyanine with 2-propoxy substituents exhibited relatively low toxicity in Vibrio fischeri bioluminescence test, whereas phthalocyanine with benzyloxy substituents revealed intense bioluminescence, which could be associated with hormesis phenomenon., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The influence of ionising radiation on the physicochemical properties of meropenem trihydrate in solid state was studied for doses of e-beam radiation: 25 kGy and 400 kGy. In the first part of our studies, we evaluated the possibility of applying radiosterilization to obtain sterile meropenem. No changes for meropenem irradiated with a dose of 25 kGy, the dose required to attain sterility, was confirmed in the results of spectroscopic (FT-IR), thermal (DSC, TGA) and X-ray powder diffraction (XRPD) studies. The radiation dose of 25 kGy produces no more than about 1500 ppm of radical defects. The chromatographic studies of irradiated meropenem in solutions did not show any chemical degradation. Moreover, the antimicrobial activity of meropenem irradiated with the dose of 25 kGy was unchanged. Based on the received results, we can conclude that radiostelization is a promising, alternative method for obtaining sterile meropenem. In the second part of the research, meropenem was exposed to e-beam radiation at the 400 kGy dose rate. It was confirmed, that reducing of antimicrobial activity could be connected with the degradation of β-lactam ring and changes in the trans-hydroxyethyl group. Apart from chemical changes, changes in the physical stability of irradiated meropenem (400 kGy) was also observed.

Zinc(II) phthalocyanine bearing eight non-peripheral 2-propoxy substituents was subjected to physicochemical study and, after incorporation in lipid vesicles, assessed as a potential photosensitizer for antibacterial photodynamic therapy. The phthalocyanine derivative obtained in the macrocyclization reaction was characterized by MS and NMR techniques. Moreover, its chemical purity was confirmed by HPLC analysis. X-ray structural analysis revealed that overcrowding of the phthalocyanine derivative leads to a strong out-of-plane distortion of the π-system of the macrocycle core. In the UV-Vis absorption spectra of zinc(II) phthalocyanine two characteristic bands were found: the Soret (300-450 nm) and the Q band (600-800 nm). Photophysical properties of mono- and diprotonated forms of phthalocyanine derivative were studied with time-resolved fluorescence spectroscopy. Its tri- and tetraprotonated forms could not be obtained, because compound decomposes in higher acid concentrations. The presented zinc(II) phthalocyanine showed values of singlet oxygen generation Φ Δ  = 0.18 and 0.16, the quantum yield of the photodecomposition Φ P  = 3.06∙10 -4 and 1.23∙10 -5 and the quantum yield of fluorescence Φ FL  = 0.005 and 0.004, designated in DMF and DMSO, respectively. For biological studies, phthalocyanine has been incorporated into modified liposome vesicles containing ethanol. In vitro bacteria photoinactivation study revealed no activity against Escherichia coli and 5.7 log reduction of the Enterococcus faecalis growth., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The crystal structures of three ester derivatives of glycyrrhetinic acid (GE) are reported. X-ray crystallography revealed that despite differences in the size of the ester substituents (ethyl, isopropyl and 2-morpholinoethyl) the scheme of molecular self-assembly is similar in all three cases but differs significantly from that observed in other known GE esters. According to our analysis, the two basic patterns of self-assembly of GE esters observed in their unsolvated crystals correspond to two distinct orientations of the ester groups relative to the triterpene backbone. Moreover, comparison of the self-assembly modes of GE esters in their unsolvated forms with the supramolecular organization of GE and carbenoxolone in their solvated crystals revealed that ester substituents replace solvent molecules hydrogen bonded to the COOH group at the triterpene skeleton, resulting in similar packing arrangements of these compounds.

Three 2-(morpholin-4-yl)ethoxy substituted phthalocyanines were synthesized and characterized. Phthalocyanine derivatives revealed moderate to high quantum yields of singlet oxygen production depending on the solvent applied (e.g., in DMF ranging from 0.25 to 0.53). Their photosensitizing potential for photodynamic therapy was investigated in an in vitro model using cancer cell lines. Biological test results were found particularly encouraging for the zinc(II) phthalocyanine derivative possessing two 2-(morpholin-4-yl)ethoxy substituents in nonperipheral positions. Cells irradiated for 20 min at 2 mW/cm(2) revealed the lowest IC50 value at 0.25 μM for prostate cell line (PC3), whereas 1.47 μM was observed for human malignant melanoma (A375) cells. The cytotoxic activity in nonirradiated cells of novel phthalocyanine was found to be very low. Moreover, the cellular uptake, localization, cell cycle, apoptosis through an ELISA assay, and immunochemistry method were investigated in LNCaP cells. Our results showed that the tested photosensitizer possesses very interesting biological activity, depending on experimental conditions.

Carbenoxolone (CBXH2), a pharmaceutically relevant derivative of glycyrrhetinic acid, was studied by X-ray crystallography. The crystal structures of its unsolvated form, propionic acid and dimethoxyethane solvates and a solvated cocrystal of the free acid with its monobasic sodium salt CBXH2·CBXHNa·(butan-2-one)2·2H2O reveal that the recurring motif of supramolecular architecture in all crystal forms is a one-dimensional ribbon with closely packed triterpene fragments. It does not result from strong specific interactions but solely from van der Waals interactions. The ribbons are further arranged into diverse layer-type aggregates with a hydrophobic interior (triterpene skeletons) and hydrophilic surfaces covered with carboxylic/carboxylate groups. Solvent molecules included at the interface between the layers influence hydrogen-bonding interactions between the carbenoxolone molecules and organization of the ribbons within the layer. Comparison of crystal structures of carbenoxolone, glycyrrhizic acid and its aglycone-glycyrrhetinic acid have shown the impact of the size and hydrophilic character of the substituent at the triterpene C3 atom on the supramolecular architecture of these three closely related molecules.

Four novel magnesium(II) and zinc(II) phthalocyanines bearing 1,4,7-trioxanonyl, polyether and/or (2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy, heterocyclic substituents at their non-peripheral positions were synthesized and assessed in terms of physicochemical and biological properties. Magnesium phthalocyanine derivatives bearing polyether substituents (Pc-1), a mixed system of polyether and heterocyclic substituents (Pc-3), and four heterocyclic substituents (Pc-4), respectively, were synthesized following the Linstead macrocyclization reaction procedure. Zinc phthalocyanine (Pc-2) bearing polyether substituents at non-peripheral positions was synthesized following the procedure in n-pentanol with the zinc acetate, and DBU. Novel phthalocyanines were purified by flash column chromatography and characterized using NMR, MS, UV-Vis and HPLC. Moreover, two precursors in macrocyclization reaction phthalonitriles were characterized using X-ray. Photophysical properties of the novel macrocycles were evaluated, including UV-Vis spectra analysis and aggregation study. All macrocycles subjected to singlet oxygen generation and the oxidation rate constant measurements exhibited lower quantum yields of singlet oxygen generation in DMSO than in DMF. In addition, the Pc-2 molecule was found to be the most efficient singlet oxygen generator from the group of macrocycles studied. The photocytotoxicity evaluated on the human oral squamous cell carcinoma cell line, HSC-3, for Pc-3 was significantly higher than that for Pc-1, Pc-2, and Pc-4. Interestingly, Pc-3 was found to be the most active macrocycle in vitro although its ability to generate singlet oxygen was significantly lower than those of Pc-1 and Pc-2. However, attempts to encapsulate phthalocyanines Pc-1-Pc-3 in liposomal membranes were unsuccessful. The phthalocyanine-nitroimidazole conjugate, Pc-4 was encapsulated in phosphatidylglycerol:phosphatidylcholine unilamellar liposomes and subjected to photocytotoxicity study., (© 2013.)

Rifampicin belongs to the family of naphthalenic ansamycin antibiotics. The first crystal structure of rifampicin in the form of the pentahydrate was reported in 1975 [Gadret, Goursolle, Leger & Colleter (1975). Acta Cryst. B 31, 1454-1462] with the rifampicin molecule assumed to be neutral. Redetermination of this crystal structure now shows that one of the phenol -OH groups is deprotonated, with the proton transferred to a piperazine N atom, confirming earlier spectroscopic results that indicated a zwitterionic form for the molecule, namely (2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)-21-acetyloxy-6,9,17,19-tetrahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[(E)-N-(4-methylpiperazin-4-ium-1-yl)formimidoyl]-1,11-dioxo-1,2-dihydro-2,7-(epoxypentadeca[1,11,13]trienimino)naphtho[2,1-b]furan-5-olate pentahydrate, C(43)H(58)N(4)O(12)·5H(2)O. The molecular structure of this antibiotic is stabilized by a system of four intramolecular O-H···O and N-H···N hydrogen bonds. Four of the symmetry-independent water molecules are arranged via hydrogen bonds into helical chains extending along [100], whereas the fifth water molecule forms only one hydrogen bond, to the amide group O atom. The rifampicin molecules interact via O-H···O hydrogen bonds, generating chains along [001]. Rifampicin pentahydrate is isostructural with recently reported rifampicin trihydrate methanol disolvate.

Porphyrazines possessing non-coordinating alkyl (propyl) and aralkyl (4-tert-butylphenyl) groups in the periphery were studied as optical sensors for a set of mono-, di- and trivalent cations. Investigated porphyrazines in the UV-Vis monitored titrations revealed significant responses towards aluminium and gallium cations, unlike other metal ions studied. Additionally, porphyrazine possessing 4-tert-butylphenyl peripheral substituents showed sensor property towards ruthenium cation and was chosen for further investigation. The presence of isosbestic points in absorption spectra for its titration with aluminium, gallium and ruthenium cations, accompanied by a linear Benesi-Hildebrand plot, proved complex formation. The continuous variation method was used to determine binding stoichiometry in 1:1 porphyrazine-metal ratio. X-Ray studies and density functional theory calculations were employed to investigate octa(4-tert-butylphenyl)porphyrazine structure. The results helped to explain the observed selectivity towards certain ions. Interaction between ion and porphyrazine meso nitrogen in a Lewis acid-Lewis base manner is proposed.

Abstract: An earlier developed alkylating path leading to tetraalkylated diaminomaleonitrile derivatives was explored. Attempts to explain the reactivity of the representative dialkylated diaminomaleonitrile 2,3-bis[(3-pyridylmethyl)amino]-2( Z )-butene-1,4-dinitrile during the alkylation reaction were performed using X-ray and density functional theory (DFT) studies. The condensed Fukui functions accompanied by softness indices were found to be useful in explaining its reactivity observed during the reaction. The values of the Fukui functions and condensed softness for electrophilic attack calculated from Mulliken, Löwdin, and natural population analyses closely corresponded to the experimental observations. When 2,3-bis[(3-pyridylmethyl)amino]-2( Z )-butene-1,4-dinitrile disodium salt was treated with dimethyl sulfate at lower temperatures the alkylation reaction prevailed, whereas at higher temperatures the alkylating agent acted as a hydride anion acceptor, which favored the elimination reaction. The tetraalkylated dinitrile 2,3-bis[methyl(3-pyridylmethyl)amino]-2( Z )-butene-1,4-dinitrile was used in the synthesis of tribenzoporphyrazine bearing methyl(3-pyridylmethyl)amino groups, which was subsequently subjected to solvatochromic and metallation studies. The changes observed during metallation seem to result from the coordination of the 3-pyridyl group by a palladium ion. This could influence the configuration of the methyl(3-pyridylmethyl)amino moiety, causing more effective donation of a lone pair of electrons from peripheral nitrogen to the macrocyclic ring., Graphical Abstract: .

CMTI-I, a small-protein trypsin inhibitor, has been crystallized as a 4:1 protein-zinc complex. The metal is coordinated in a symmetric tetrahedral fashion by glutamate/glutamic acid side chains. The structure was solved by direct methods in the absence of prior knowledge of the special position metal centre and refined with anisotropic displacement parameters using diffraction data extending to 1.03 A. In the final calculations, the main-chain atoms of low B(eq) values were refined without restraint control. The two molecules in the asymmetric unit have a conformation that is very similar to that reported earlier for CMTI-I in complex with trypsin, despite the Met8Leu mutation of the present variant. The only significant differences are in the enzyme-binding epitope (including the Arg5 residue) and in a higher mobility loop around Glu24. The present crystal structure contains organic solvent molecules (glycerol, MPD) that interact with the inhibitor molecules in an area that is at the enzyme-inhibitor interface in the CMTI-I-trypsin complex. A perfectly ordered residue (Ala18) has an unusual Ramachandran conformation as a result of geometrical strain introduced by the three disulfide bridges that clamp the protein fold. The results confirm deficiencies of some stereochemical restraints, such as peptide planarity or the N-C(alpha)-C angle, and suggest a link between their violations and hydrogen bonding.

Enolase is a metalloenzyme which catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP). Mg2+ and Zn2+ are cofactors which strongly bind and activate the enzyme. Ca2+ also binds strongly but does not produce activity. Phosphoglycolate (PG) is a competitive inhibitor of enolase. The structures of two inhibitory ternary complexes: yeast enolase-Ca2(+)-PGA and yeast enolase-Zn2(+)-PG, were determined by X-ray diffraction to 2.2-A resolution and were refined by crystallographic least-squares to R = 14.8% and 15.7%, respectively, with good geometries of the models. These structures are compared with the structure of the precatalytic ternary complex enolase-Mg2(+)-PGA/PEP (Lebioda & Stec, 1991). In the complex enolase-Ca2(+)-PGA, the PGA molecule coordinates to the Ca2+ ion with the hydroxyl group, as in the precatalytic complex. The conformation of the PGA molecule is however different. In the active complex, the organic part of the PGA molecule is planar, similar to the product. In the inhibitory complex, the carboxylic group is in an orthonormal conformation. In the inhibitory complex enolase-Zn2(+)-PG, the PG molecule coordinates with the carboxylic group in a monodentate mode. In both inhibitory complexes, the conformational changes in flexible loops, which were observed in the precatalytic complex, do not take place. The lack of catalytic metal ion binding suggests that these conformational changes are necessary for the formation of the catalytic metal ion binding site.

Alcohol oxidase from Pichia pastoris has been crystallized from polyethylene glycol 4000 solutions. The crystals are tetragonal, a = 228 A, c = 456 A space group P4(1)2(1)2. The crystals scatter only to about 6 A resolution; their poor crystallinity may have some physiological function. Secondary structure predictions suggest that the C-terminal part of the molecule, residues 311-664, has the folding of an eightfold beta/alpha-barrel (TIM barrel). This would indicate common ancestry with four other flavoenzymes: canavalin, glycolate oxidase, flavocytochrome b, and trimethylamine dehydrogenase.