Characterization of Ambroxol and Its Hydrochloride Salt Crystals by Bromine K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and X-Ray Crystal Structure Analysis.

Polymorphic crystals of ambroxol, forms I and II, and form A ambroxol hydrochloride crystals were characterized with bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy and single-crystal X-ray structure analysis. The XANES spectra had unique shapes depending on the crystal forms. Refined single-crystal structures revealed different interatomic interactions around bromine atoms, such as C-H…Br and N-H…Br hydrogen bonds, Br…O halogen bonds, and N-H…π interactions. Differences in these weak interactions could affect the electronic states of the bromines, resulting in differences in the XANES spectra. The results demonstrated that weak non-conventional interatomic interactions could alter the shape of XANES spectra. Hence, the spectra could be used for evaluating polymorphs of active pharmaceutical ingredients.

Novel Pharmaceutical Cocrystals and Solvate Crystals of Nobiletin, a Citrus Flavonoid with Potent Pharmacological Activity.

Nobiletin (NOB) is a flavonoid with attractive pharmaceutical characteristics, including anti-Alzheimer's, anti-inflammation, and anti-cancer properties, but it has low solubility in water, resulting in reduced bioavailability. Its solubility must be improved to develop NOB as a drug. Cocrystal engineering can change the physicochemical properties of an active pharmaceutical ingredient and generate remarkable drug candidates that are superior in drug formulation. In this report, extensive co-crystal screening of NOBs with 31 cocrystal formers (coformers) with various functional groups was carried out by the liquid-assisted grinding method. As a result, four cocrystals (NOB with urea (URE), oxalic acid, gallic acid and salicylic acid) and one solvate crystal (NOB with formic acid (FOR)) were found. Powder X-ray diffraction and thermal analysis revealed the unique crystal morphology of all the obtained samples. In addition, the crystal structures of two of them (NOB-URE and NOB-FOR) were determined by single crystal X-ray diffraction. The results revealed that NOB-URE and NOB-FOR are new cocrystals or solvate crystals consisting of molar ratios of 1 : 2 and 1 : 0.73, respectively. In NOB-URE, we could observe a transient increase in solubility due to supersaturation, suggesting that URE is one of the better coformers of NOB.

Comparison of the physical properties of disodium etidronate amorphous forms prepared by different manufacturing methods.

Amorphous forms of disodium etidronate were prepared by three manufacturing methods, heat drying, freeze drying, and anti-solvent precipitation, and the effects of these methods on the physical properties of disodium etidronate amorphous forms were evaluated for the first time. Variable temperature X-ray powder diffraction and thermal analyses revealed that these amorphous forms had different physical properties such as glass transition point, water desorption, and crystallization temperatures. These differences can be explained by the molecular mobility and water content in amorphous forms. The differences in the structural characteristics related to the differences in these physical properties could not be detected clearly by the spectroscopic methods like Raman spectroscopy and X-ray absorption near-edge spectroscopy. Dynamic vapor sorption analyses demonstrated that all amorphous forms were hydrated to form I, a tetrahydrated form, at above 50% relative humidity, and the transition to form I was irreversible. These amorphous forms require strict humidity control to avoid crystallization. Among the three amorphous forms of disodium etidronate, the amorphous form prepared by heat drying was the most suitable for manufacturing the solid formulation, considering the low water content and low molecular mobility.

Analysis of Cimetidine Crystal Polymorphs by X-ray Absorption Near-Edge Spectroscopy.

Sulfur K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of the active pharmaceutical ingredients (APIs) containing sulfur atoms. Cimetidine (CIM) was used as a model API. Each crystal form of CIM has its own XANES spectrum, so we can discriminate the crystal form by its spectrum. The analysis of the crystal structure of CIM revealed that the difference in the shape of XANES spectra was ascribable to the difference in the C-S-C bond angle of CIM molecules and the intermolecular hydrogen bonds, such as C-H···S and N-H···S, and S-S interaction. It was found that the peak shape of the XANES spectrum is gentle when the C-S-C bond angle is large, while the peak shape can be steep when the C-S-C bond angle is small. Furthermore, it was found that the peak energy values varied depending on the hydrogen bonds and S-S interaction. By linear combination fitting using XANES spectra, it was possible to quantify the ratio of CIM form A crystal in mixed powders of form A and monohydrate crystals. These results indicate that XANES measurements can be a useful technique to evaluate the crystal polymorphism of APIs containing S atom in pharmaceutical formulation.

C-H▪▪▪S Hydrogen Bonds in Ampicillin and Amoxicillin Crystals Investigated by Sulfur K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy and Single-Crystal X-Ray Structure Analysis.

Sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was evaluated for its ability to detect non-conventional C-H▪▪▪S hydrogen bonds in crystals of the sulfur-containing penam antibiotics ampicillin and amoxicillin. The XANES spectra of the nearly isomorphous crystals of ampicillin trihydrate and amoxicillin trihydrate were very similar, whereas that of ampicillin anhydrate displayed unique features. Single-crystal X-ray structure analyses revealed that the C-H▪▪▪S hydrogen bond geometries and the chemical types of the hydrogen donors differed between the isomorphous trihydrate crystals and ampicillin anhydrate crystal. These observations demonstrate that the shapes of the sulfur K-edge XANES spectra are dependent on the nature of the C-H▪▪▪S hydrogen bonds. Sulfur K-edge XANES spectroscopy shows promise for use in the detection and analysis of non-covalent interactions, including hydrogen bonds to sulfur atoms, within active pharmaceutical ingredients.

Direct detection of the crystal form of an active pharmaceutical ingredient in tablets by X-ray absorption fine structure spectroscopy.

Different crystal forms of active pharmaceutical ingredients (APIs) may display variations in physicochemical properties. During the drug development process, the definitive purpose is to maintain homogeneous quality in a single crystalline form. Hence, it is important to evaluate and understand the properties of each crystal form of APIs in pharmaceutics. In this study, forms 0, Ⅰ, Ⅱ, III of bromhexine hydrochloride, and form S of bromhexine were characterized by the commonly used methods X-ray powder diffraction, thermogravimetry-differential thermal analysis, and single crystal structure X-ray diffraction. Additionally, X-ray absorption fine structure spectroscopy (XAFS), a seldom used method in the pharmaceutics discipline was also applied to explore the chemical environment of bromine atoms in forms 0, Ⅰ, Ⅱ and S as well as chloride ions in forms 0 to Ⅱ. The XAFS spectra of each form were different from each of the other forms which indicated the chemical environment around target elements in the crystal polymorphs were distinct. Then, we measured the commercial bromhexine hydrochloride tablets with XAFS measurement and found that XAFS could distinguish the crystal form in the tablets. Hence, we demonstrated that XAFS measurements would be applicable as one of the methods for the direct detection of APIs in the tablets.

Bromine K-Edge X-Ray Absorption Near-Edge Structure Analysis on Hydrobromide-Salt Crystals and the Solid Dispersion of Active Pharmaceutical Ingredients.

Bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy analyses were used to evaluate the crystals of the active pharmaceutical ingredients, eletriptan hydrobromide, dextromethorphan hydrobromide and scopolamine hydrobromide salts and the solid dispersion of eletriptan hydrobromide. The crystals and the solid dispersion of the active pharmaceutical ingredient (API) salts could be discriminated based on the shape of the XANES spectra. The differences in the shape of XANES spectra was ascribable to the differences in the interatomic interactions of the bromine ions based on the crystal structures. Ratio of the eletriptan hydrobromide α-form crystal in mixed powders of α-form and monohydrate crystals could be quantified by the linear-combination fitting using their XANES spectra. These results indicated that the XANES spectroscopy are a potent method for evaluating the APIs of pharmaceutical formulations even at the higher energy region around the bromine K-edge of 13470 eV.

Investigation of Physical Properties of Disodium Etidronate Tetrahydrate and Application of Phosphorus K-Edge X-Ray Absorption Near-Edge Structure Spectroscopy.

PURPOSE: Disodium etidronate is a bisphosphonate, compounds that are widely used in the treatment of bone disorders such as osteoporosis and Paget's disease. We investigated the physical properties of disodium etidronate tetrahydrate crystal, form I. METHODS: We used X-ray powder diffraction (XRPD), thermal analysis, dynamic vapor sorption (DVS), X-ray single crystal structure analysis, and phosphorus K-edge X-ray absorption near-edge structure (XANES) spectroscopy for the first time. RESULTS: XRPD and thermal analyses demonstrated that form I was dehydrated and transformed to an amorphous form, to a crystalline form II, and finally to a form III by heating. DVS measurements revealed that the amorphous form, form II, and form III were rehydrated to form I by humidification, and form I was stable even at 0% relative humidity. These results indicate that form I is the most stable solid-state under ambient conditions and is suitable as an API for manufacture in solid formulations. The phosphorus K-edge XANES spectra differed among form I, the amorphous form, and form II, which may be ascribed to the difference in the coordinate bond schemes between the phosphate moieties and sodium ions. The results demonstrated that the phosphorus K-edge XANES spectroscopy could be applied to the identification or the discrimination of crystal forms of the APIs containing phosphate moieties. CONCLUSIONS: Acquired information about physical properties are crucial for manufacturing of solid formulations of disodium etidronate. XANES spectroscopy is a promising alternative method for evaluating the solid-state forms of APIs.

X-ray Absorption Near-Edge Spectroscopy Analysis of Indomethacin in Crystalline Forms and in Amorphous Solid Dispersions.

Chlorine K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of identical active pharmaceutical ingredients (APIs) containing chloride atoms and their amorphous solid dispersions (ASDs). Indomethacin (IMC), of which three crystal forms (α, ß, and γ) have been reported, was used as a model API. The shape of XANES spectra was unique to each IMC crystal. The analysis of the crystal structures of IMC revealed that chlorine atoms of the IMCα form had unique intermolecular interactions and halogen bonds with oxygen atoms, while those of the IMCγ form do not have any notable interactions. This result showed that XANES measurements can detect weak interatomic interactions. The shapes of the ASD spectra were clearly different from those of the crystals, suggesting that the environment around the Cl atom of IMC was different from that of the crystals. A thermal stress test was then performed to study the transformation from the amorphous form to the crystalline form of IMC in the ASD. The powder X-ray diffraction (PXRD) patterns indicated that amorphous IMC transformed into crystals during the thermal stress test. In accordance with the PXRD results, the XANES spectra also transformed from ASD to crystalline form. These results indicate that the IMC transformation could be monitored by XANES measurement. Our findings led us to conclude that XANES measurement is a novel approach for the evaluation of crystal polymorphs of APIs and the crystalline state of APIs in ASDs.

Reduced deliquescency of isosorbide by cocrystallization and mechanisms for hygroscopicity.

Isosorbide (ISO) is an effective hyperosmotic agent that can be administrated orally and is used as a therapeutic agent for brain pressure drop, glaucoma, and Meniere's disease. However, the critical relative humidity (CRH) of ISO is about 48% RH at 25 °C, and it deliquesces in humid environments. In this study, we attempted to reduce the deliquescence of ISO using cocrystallization and analyze the water adsorption mechanism from the crystal structure. Four new ISO cocrystals with piperazine (PZ), hydrochlorothiazide (HCT), 3,5-dihydroxybenzoic acid (35DHBA), or gallic acid (GA) were identified. The dynamic vapor sorption analyses demonstrated that all the cocrystals showed higher CRHs than the ISO crystal. Although water adsorption below the CRH was observed for all cocrystals, the water molecules adsorbed in the ISO-PZ and ISO-GA cocrystals were lower than those in the ISO crystal. Investigation of the crystal structures suggested that the amount of water adsorbed might be related to the degree of exposure of the ISO hydroxyl groups on the crystal surface. Given the CRH, water adsorption below the CRH, thermal stability, apparent dissolution rate, and toxicity level of the coformer, the ISO-GA cocrystal is the most suitable for preparing a solid formulation of ISO.

Chlorine K-Edge X-Ray Absorption Near-Edge Structure Analysis of Clarithromycin Hydrochloride Metastable Crystal.

X-ray absorption fine structure (XAFS) spectra, especially X-ray absorption near-edge structure (XANES), show unique features depending on the chemical states and structures in the vicinity of the X-ray absorbing elements. As such, they can be used to identify the chemical environment of elements. XAFS spectroscopy was applied to investigate the chemical environment of chloride ions in a metastable form A crystal of the antibiotic clarithromycin hydrochloride hydrate. The XANES of the form A crystals showed low similarity to that of the active pharmaceutical ingredient (API) hydrochloride salt crystals, but showed a high similarity to that of a hydrochloride aqueous solution. This indicated that the chloride ion in the form A crystals predominantly interacted with water molecules and was fully hydrated, which was consistent with the previous presumption that form A may be high water-content crystals. This study demonstrated that this XAFS spectroscopy method would be a potent alternative technique for evaluating APIs.

Correlation between drug dissolution and resistance to water-induced phase separation in solid dispersion formulations revealed by solid-state NMR spectroscopy.

We aimed to elucidate the dissolution mechanism of solid dispersions (SDs) according to the carrier polymers used. Nifedipine (NIF) and polymers dissolved simultaneously from NIF/Eudragit® S (EUD-S), NIF/Eudragit® L (EUD-L), and NIF/hypromellose (HPMC)/EUD-S spray-dried samples (SPDs). In contrast, NIF dissolved separately from polymers from NIF/HPMC and NIF/HPMC/EUD-L SPDs due to the formation of an amorphous NIF-rich interface. Solid-state NMR spectroscopy indicated that NIF-EUD interactions were stronger than NIF-HPMC interactions. NIF/HPMC SPD exhibited weak interactions; thus, it failed to inhibit phase separation during the dissolution process and control NIF dissolution. The hygroscopicity of SPDs was higher with HPMC mixing and increased substitution ratio of methacrylic acid in EUD. Moreover, solid-state NMR spectroscopy revealed that the NIF-EUD interactions were hindered to a large extent by the absorbed water. During the dissolution process of NIF/HPMC/EUD-L SPD, the introduction of water to the NIF-EUD-L interaction site could induce the phase separation and poor controllability of NIF dissolution. Water-induced phase separation should be considered based on molecular-level characterization to obtain SDs with enhanced drug dissolution. An investigation of the molecular state change caused by the absorbed water using solid-state NMR spectroscopy will be helpful in understanding the dissolution mechanism of SDs.

Structural and biochemical basis of the formation of isoaspartate in the complementarity-determining region of antibody 64M-5 Fab.

The formation of the isoaspartate (isoAsp) is one of spontaneous degradation processes of proteins, affecting their stability and activity. Here, we report for the first time the crystal structures of an antibody Fab that contains isoAsp in the complementarity-determining region (CDR), along with biochemical studies to detect isoAsp. By comparing the elution profiles of cation-exchange chromatography, it was clarified that the antibody 64M-5 Fab is converted from the normal form to isoAsp form spontaneously and time-dependently under physiological conditions. The isoAsp residue was identified with tryptic peptide mapping, N-terminal sequencing, and the protein isoaspartyl methyltransferase assay. Based on the fluorescence quenching method, the isoAsp form of 64M-5 Fab shows a one order of magnitude lower binding constant for its dinucleotide ligand dT(6-4)T than the normal form. According to the structure of the isoAsp form, the conformation of CDR L1 is changed from the normal form to isoAsp form; the loss of hydrogen bonds involving the Asn28L side-chain, and structural conversion of the ß-turn from type I to type II'. The formation of isoAsp leads to a large displacement of the side chain of His27dL, and decreased electrostatic interactions with the phosphate group of dT(6-4)T. Such structural changes should be responsible for the lower affinity of the isoAsp form for dT(6-4)T than the normal form. These findings may provide insight into neurodegenerative diseases (NDDs) and related diseases caused by misfolded proteins.

Crystal Structures of Flavone C-Glycosides from Oolong Tea Leaves: Chafuroside A Dihydrate and Chafuroside B Monohydrate.

Chafuroside A and chafuroside B are flavone C-glycosides isolated from oolong tea leaves. They have a number of beneficial pharmacological activities related to antiinflammation at various concentrations. However, no crystallographic study of chafurosides has yet been reported. In the present study, the crystal structures of chafuroside A and chafuroside B were investigated using single-crystal X-ray diffraction. The asymmetric unit of the chafuroside A crystal consists of one chafuroside A and two water molecules, and that of chafuroside B contains one chafuroside B and one water molecule. The flavone moiety of chafuroside A is curved, i.e., the angle between the best-fit planes of the chromene and phenyl rings is 18.9°, whereas the chafuroside B flavone moiety is relatively flat. A comparison of the curvatures of the flavone moieties of various C-glycosides showed that the curvature of chafuroside A is significantly larger than those of the others. This structural feature might contribute to the differences between the strengths of the pharmacological activities of chafurosides A and B.

Desolvation behavior of indinavir sulfate ethanol and follow-up by terahertz spectroscopy.

Active pharmaceutical ingredients are composed of single-component or multicomponent crystals. Multicomponent crystals include salts, co-crystals, and solvates. Indinavir sulfate is the ethanol solvate form of indinavir that is known to deliquesce through moisture absorption. However, the detailed behavior of solvent molecules in the crystal has not been investigated. In this study, we studied the desolvation mechanism of indinavir sulfate ethanol and investigated the behavior of solvent molecules in the solid from. Indinavir sulfate ethanol contained 1.7 molecules of ethanol, 0.7 of which desolvated at room temperature. They were originally two ethanol solvent molecules; one molecule of ethanol desolvated at room temperature, and the conformation of the remaining ethanol and t-butyl groups changed in conjunction with the removal of one ethanol molecule. Desolvation could hardly be detected by powder X-ray diffraction; however, it was detected using terahertz spectroscopy. Terahertz measurement of desolvation showed a high correlation with thermogravimetry data, suggesting that desolvation could be observed non-destructively using terahertz spectroscopy. We concluded that indinavir sulfate 1 ethanol deliquesced at 60% relative humidity, and it turned into an amorphous solid after drying.

Structures of the antibody 64M-5 Fab and its complex with dT(6-4)T indicate induced-fit and high-affinity mechanisms.

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts produced by ultraviolet light are mutagenic and carcinogenic. The crystal structures of the anti-(6-4) photoproduct antibody 64M-5 Fab and of its complex with dT(6-4)T were determined at 2.5 and 2.0 Šresolution, respectively. A comparison between the dT(6-4)T-liganded and unliganded structures indicates that the side chain of His93L is greatly rotated and shifted on binding to dT(6-4)T, leading to the formation of an electrostatic interaction with the phosphate moiety of dT(6-4)T, which shows a remarkable induced fit. Based on a comparison of the dT(6-4)T-liganded structures of the 64M-5 and 64M-2 Fabs, the electrostatic interaction between the side chain of His93L in 64M-5 and the phosphate moiety of dT(6-4)T is lost for Leu93L in 64M-2, while Arg90L in 64M-5 instead of Gln90L in 64M-2 stabilizes the conformation of complementarity-determining region (CDR) L3. These differences contribute to the higher affinity of 64M-5 for dT(6-4)T compared with that of 64M-2.

Mechanism of Drug Release From Temperature-Sensitive Formulations Composed of Low-Melting-Point Microcrystalline Wax.

It was reported that wax matrix (WM) particles composed of low-melting-point microcrystalline wax showed unique release behaviors; the particles released only a small amount of the entrapped drug (non-diffusion-controlled release) at 37°C, whereas it showed comparatively fast drug release in a diffusion-controlled manner at 25°C. However, the mechanism of the drug release is still unclear. The objective of this study was to determine the mechanism of drug release from the WM particles using X-ray computed tomography. In the WM particles collected during dissolution tests at 25°C, the void space derived from drug release increased with increasing time, and there was no change in the structure, indicating that the WM particles released drug while maintaining the particle shape at 25°C. In the WM particles collected during dissolution tests at 37°C, the void space was confirmed at initial time point; however, at subsequent time points, the void space was disappeared, and the roughness of the surface was evident. This structural change may have blocked the conveyance pathway of the outer medium, which would inhibit the drug release. The difference between the drug-release mechanisms of the WM particles at the 2 temperatures will be valuable for developing cooling-triggered, temperature-sensitive formulations.

Phase transitions of antibiotic clarithromycin forms I, IV and new form VII crystals.

Metastable crystal form I of the antibiotic clarithromycin has a pharmaceutically valuable characteristic that its crystalline phase transition can be applied for its sustained release from tablets. The phase transition of form I was investigated in detail by single crystal and powder X-ray analyses, dynamic vapor sorption analysis and thermal analysis. The single crystal structure of form I revealed that form I was not an anhydrate crystal but contained a partially occupied water molecule in the channel-like void space. Dynamic vapor sorption (DVS) analysis demonstrated that form I crystals reversibly sorbed water molecules in two steps when the relative humidity (RH) increased and finally transited to hydrate form IV at 95% RH. DVS analysis also showed that when the RH decreased form IV crystals lost water molecules at 40% RH and transited to the newly identified anhydrate crystal form VII. Form VII reversibly transited to form IV at lower RH than form I, suggesting that form I is more suitable for manufacturing a sustained-release tablet of CAM utilizing the crystalline phase transition.

Stabilization Mechanism of Roxithromycin Tablets Under Gastric pH Conditions.

Macrolide antibiotics are widely used at clinical sites. Clarithromycin (CAM), a 14-membered macrolide antibiotic, was reported to gelate under acidic conditions. Gelation allows oral administration of acid-sensitive CAM without enteric coating by hindering the penetration of gastric fluid into CAM tablets. However, it is unknown whether this phenomenon occurs in other macrolide antibiotics. In this study, we examined the gelation ability of 3 widely used macrolide antibiotics, roxithromycin (RXM), erythromycin A, and azithromycin. The results indicated that not only CAM but also RXM gelated under acidic conditions. Erythromycin A and azithromycin did not gelate under the same conditions. Gelation of RXM delayed the disintegration of the tablet and release of RXM from the tablet. Disintegration and release were also delayed in commercial RXM tablets containing disintegrants. This study showed that 2 of the 4 macrolides gelated, which affects tablet disintegration and dissolution and suggests that this phenomenon might also occur in other macrolides.

Mechanism of the formation of hollow spherical granules using a high shear granulator.

Recently, we have developed a novel granulation technology to manufacture hollow spherical granules (HSGs) for controlled-release formulations; however, the mechanism of the granulation is still unclear. The aim of this study is to determine the mechanism of the formation of the HSGs using a high shear granulator. Samples of granulated material were collected at various times during granulation and were investigated using scanning electron microscope and X-ray computed tomography. It was observed that the granulation proceeded by drug layering to the polymer, followed by formation of a hollow in the granule. In addition, it was also found that generation of a crack in the adhered drug layer and air flow into the granules might be involved in forming the hollow in the structure. Observation of the granulation of formulations with different types of drugs and polymers indicated that negative pressure in the granules occurred and the granules caved in when the hollow was formed. The hollow-forming speed and the shell density of the hollow granules depended on the particular drug and polymer. Taken together, the granulation mechanism of HSGs was determined and this information will be valuable for HSGs technology development.