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BACKGROUND: Internet-cognitive behavioural therapy (iCBT) for depression can include multiple components. This study explored depressive symptom improvement prognostic factors (PFs) and effect modifiers (EMs) for five common iCBT components including behavioural activation, cognitive restructuring, problem solving, self-monitoring, and assertion training. METHODS: We used data from a factorial trial of iCBT for subthreshold depression among Japanese university students (N=1093). The primary outcome was the change in PHQ-9 scores at 8weeks from baseline. Interactions between each component and various baseline characteristics were estimated using a mixed-effects model for repeated measures. We calculated multiplicity-adjusted p-values at 5% false discovery rate using the Benjamini-Hochberg procedure. RESULTS: After multiplicity adjustment, the baseline PHQ-9 total score emerged as a PF and exercise habits as an EM for self-monitoring (adjusted p-values

BackgroundInternet-based cognitive-behavioural therapy (iCBT) is effective for subthreshold depression. However, which skills provided in iCBT packages are more effective than others is unclear. Such knowledge can inform construction of more effective and efficient iCBT programmes.ObjectiveTo examine the efficacy of five components of iCBT for subthreshold depression.MethodsWe conducted an factorial trial using a smartphone app, randomly allocating presence or absence of five iCBT skills including self-monitoring, behavioural activation (BA), cognitive restructuring (CR), assertiveness training (AT) and problem-solving. Participants were university students with subthreshold depression. The primary outcome was the change on the Patient Health Questionnaire-9 (PHQ-9) from baseline to week 8. Secondary outcomes included changes in CBT skills.FindingsWe randomised a total of 1093 participants. In all groups, participants had a significant PHQ-9 reduction from baseline to week 8. Depression reduction was not significantly different between presence or absence of any component, with corresponding standardised mean differences (negative values indicate specific efficacy in favour of the component) ranging between −0.04 (95% CI −0.16 to 0.08) for BA and 0.06 (95% CI −0.06 to 0.18) for AT. Specific CBT skill improvements were noted for CR and AT but not for the others.ConclusionsThere was significant reduction in depression for all participants regardless of the presence and absence of the examined iCBT components.Clinical implicationWe cannot yet make evidence-based recommendations for specific iCBT components. We suggest that future iCBT optimisation research should scrutinise the amount and structure of components to examine.Trial registration numberUMINCTR-000031307.

Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) regulate the growth and morphogenesis of various exocrine glands with branched morphologies. Their roles in lacrimal gland (LG) development remain unknown, but fibroblast growth factor (FGF) 10 is crucial for early LG organogenesis. To clarify the roles of EGF, HGF, and FGF10 in LG development, LG epithelial cells were isolated from late-embryonic and neonatal mice; cultured; and treated with EGF, HGF, or FGF10 and their respective receptor tyrosine kinase (RTK) inhibitors AG1478, PHA665752, or SU5402. EGF and HGF increased the number of viable cells by enhancing DNA synthesis, FGF10 and SU5402 showed no such effect, and RTK inhibitors exhibited the opposite effect. EGF and HGF receptors were immunostained in cultured late-embryonic LG epithelial cells and terminal LG acini from late embryos and adult mice. HGF was detected in neonatal LG epithelial cell culture supernatants by western blotting. In the absence of EGF and HGF RTK inhibitors, growth factor addition increased the number of viable cells and suppressed cell death. However, when one RTK was inhibited and a growth factor targeting an intact RTK was added, the number of dead cells increased as the number of viable cells increased. No cells survived when both RTKs were inhibited. In explant cultures of LGs from embryos, AG1478 or PHA665752 decreased the number of Ki67-positive proliferating epithelial cells in terminal acini. Thus, EGF and HGF may function in a cooperative autocrine manner, supporting cell proliferation and survival during LG development in late-embryonic and neonatal mice.

Proliferative diabetic retinopathy (PDR) is a vision-threatening complication of diabetes mellitus (DM). Systemic and intraocular factors are intricately related to PDR, and vitreous fluid (VF) cytokines are representative intraocular biomarkers. However, the associations between systemic factors and VF cytokines and their influence on PDR pathology are unclear. This study aimed to examine the correlation between systemic factors and VF cytokines and analyze their contributions to the pathology of PDR using multivariate analyses. We conducted a retrospective observational study on 26 PDR eyes of 25 patients with type 2 DM, and 30 eyes of 30 patients with idiopathic macular hole or epiretinal membrane as controls. Fifteen systemic and laboratory tests including blood pressure (BP) and body mass index (BMI), and 27 cytokines in VF were analyzed. BP and BMI correlated positively with VF levels of IL-6 and IP-10 in PDR patients, while no significant correlation was found between systemic factors and VF cytokines in controls. MCP-1 and VEGF-A in VF separately clustered with different systemic factors in controls, but these cytokines lost the property similarity with systemic factors and acquired property similarity with each other in PDR. Systemic factors contributed to only 10.4%, whereas VF cytokines contributed to 42.3% out of 52.7% variance of the whole PDR dataset. Our results suggest that intraocular factors play a major role in the pathology of PDR, whereas systemic factors may have limited effects, and that BP and BMI control in PDR could be useful interventions to improve intraocular immune condition.

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.

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.

The crystal forms of active pharmaceutical ingredients must be precisely determined to properly regulate their physical properties. In this study, chlorine K-edge X-ray absorption near-edge structu...

Controlling the physicochemical properties of a drug formulation is important for proper drug efficacy, since in the gastrointestinal tract many drugs undergo dissolution, limiting their efficacy. Factors affecting a drug's physicochemical properties include its crystal habit. Therefore, we predicted the crystal habit by molecular simulation for the purpose of controlling crystal morphology. In this study, we used aspirin as a model compound. By performing simulations based on known crystal structure data, we trained the simulation algorithm to produce the cubic and plate-like morphologies of crystals actually obtained. By these methods, we showed that the crystal plane of the crystal form actually obtained coincides with the characteristic crystal plane obtained by simulation. Furthermore, to consider the influence of the crystallization solvent on crystal growth, we simulated adsorption of solvent molecules on characteristic crystal planes. The difference in adsorption energy of the solvent molecules prevents the aspirin molecules from attaching to the crystal plane. As a result, we concluded that the crystal habit was caused by the difference in growth rate of the crystal plane. By applying the methods developed in this research, the growth of crystal planes can be predicted by molecular simulation, making it possible to efficiently obtain crystal forms with optimal physical properties for drug development. We believe that further development of this approach will lead to dramatic decreases in the cost and duration of drug development.

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.

Purpose To report a case of meningeal carcinomatosis by metastasis of signet-ring cell carcinoma resulting in severe visual impairment. Case presentation A 49-year-old man visited our hospital complaining of blurred vision in his left eye since 2 months ago. There was no particular history. Best corrected visual acuity (BCVA) was 25/20 in the right eye and 20/20 in the left eye. Papillary swelling in both eyes and disc hemorrhage in the left eye were observed. Gradually, the frequency of dizziness increased, and magnetic resonance imaging (MRI) suggested ventricular enlargement, infiltration around the optic nerve, and meningeal carcinomatosis. Increased cerebrospinal fluid pressure was found, and metastatic adenocarcinoma cells were observed by cerebrospinal fluid cytology. A gastroduodenal biopsy confirmed the diagnosis of poorly differentiated adenocarcinoma with signet-ring cells. Visual acuity at the final visit was 4/200 in both eyes. Conclusion If papillary swelling is observed in both eyes, meningeal carcinomatosis should be considered as a differential diagnosis.

Irritable bowel syndrome (IBS) is one of the most common functional disorders, and an intervention targeting IBS non-patients (with symptoms and undiagnosed) is needed. This study examined the efficacy of acceptance and commitment therapy (ACT) in IBS non-patients. A total of 26 non-patients were randomly assigned to either an intervention group (n = 14) or a waiting list group (n = 12). Self-reported IBS symptom severity, quality of life, psychological distress (anxiety and depression), and psychological flexibility were assessed at three time points. The intervention group completed an ACT program consisting of a one-day group session and a two-month self-help program. This program reduced depressive mood but not symptom severity. These results suggest that this ACT program is insufficient for IBS non-patients and the intervention needs further modification.

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.

1&alpha, 25-dihydroxy-22-oxacalcitriol (maxacalcitol) is a non-calcemic vitamin D3 analog clinically approved to treat psoriasis, and its role has been increasingly recognized in suppressing keratinocyte proliferation, mediating inflammation, and regulating the immune response. A large number of studies have suggested that vitamin D plays an important role in maintaining ocular surface health. However, its topical effects on the Meibomian gland (MG) has been insufficiently investigated. Here, we introduce an experimental MG orifice injury model, where the partial glandular loss occurred after electrical cauterization on a limited number of MG orifices, and investigate the efficacy and safety of maxacalcitol ointment in treating this MG orifice injury model. We confirm the alleviation of MG atrophy and ductal dilation by maxacalcitol ointment application. The recovery of injured MG visualizing as the residual MG area is significantly better in the maxacalcitol group (p = 0.020) compared with the Vaseline&reg, group, especially during the first two weeks. The cornea and other ocular tissues were not affected by maxacalcitol ointment application during our two-month observation period. Altogether, this work indicates that maxacalcitol has therapeutic potential in the amelioration of initial injury of MG orifices caused by electrocautery.

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.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.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.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.

BackgroundNeovascular age-related macular degeneration (nAMD) is a leading cause of blindness in older people. Low-grade inflammation is well-known as one of the pathogenic mechanisms in nAMD. Anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment for nAMD, although macula atrophy (MA) developed under anti-VEGF therapy causes irreversible visual function impairment and is recognized as a serious disorder. Here, we show specific expression patterns of aqueous humor (AH) cytokines in nAMD eyes developing MA under intravitreal injection of aflibercept (IVA) as an anti-VEGF antibody and present predictive cytokines as biomarkers for the incidence of MA in nAMD eyes under IVA treatment.MethodsTwenty-eight nAMD patients received three consecutive monthly IVA, followed by a pro re nata regimen for 2 years. AH specimens were collected before first IVA (pre-IVA) and before third IVA (post-IVA). AH cytokine levels, visual acuity (VA), and central retinal thickness (CRT) were measured.ResultsTwo-year incidence of MA was 21.4%. In nAMD eyes developing MA [MA (+) group], pre-IVA levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1β, VEGF and post-IVA level of MCP-1 were higher than those in nAMD eyes without MA [MA (−) group]. In hierarchical cluster analysis, pre-IVA MCP-1 and VEGF were grouped into the same subcluster, as were post-IVA MCP-1 and CRT. In principal component analysis, principal component loading (PCL) of pre-IVA interferon-γ-inducible protein 10 (IP-10) was 0.61, but PCL of post-IVA IP-10 decreased to −0.09. In receiver operating characteristic analysis and Kaplan–Meier curves, pre-IVA MCP-1, MIP-1β, and VEGF and post-IVA interleukin-6, MCP-1, and MIP-1β were detected as predictive factors for MA incidence. In 2-year clinical course, changes of VA in groups with high levels of pre-IVA MIP-1β (over 39.9 pg/ml) and VEGF (over 150.4 pg/ml) were comparable to those in MA (+) group.ConclusionSubstantial loss of IP-10 effects and persistent inflammation contribute to incidence of MA, and screening of AH cytokine levels could be a useful method to predict MA incidence in nAMD eyes under anti-VEGF therapy.

The retinal pigment epithelium (RPE) is the major source of cytokines in the retina regulating the intraocular immune environment, and a primary target of photodamage. Here, we examined 27 types of cytokines secreted by ARPE-19 cells exposed to visible light and incubated with aflibercept or ranibizumab, which are two anti-vascular endothelial growth factor (VEGF) antibodies. The cells were cultured for 24 h in the dark or under 2000 lux irradiation from a daylight-colored fluorescent lamp, and cytokine levels in the culture supernatant were measured. In the light-irradiated culture, the levels of IL-9, IL-17A and bFGF were higher, and the levels of IL-6, IL-7, IL-8 and MCP-1 were lower than those in the dark culture, while there was no significant difference with the VEGF-A level. In subgroup analyses of the light-irradiated culture, the bFGF level under 250 to 2000 lux irradiation was elevated in a light intensity-dependent manner. In culture exposed to blue, green or red light, the bFGF level was elevated by blue light and was high compared to that by green or red light. In culture with aflibercept or ranibizumab in the dark, the levels of IL-6, IL-8, bFGF and MCP-1 were increased, and the IL-12 level decreased synchronously with a reduction in the VEGF-A level. Our findings indicate that continuous irradiation of visible light and VEGF suppression may be an influential factor in expression patterns of inflammatory cytokines secreted by human RPE cells.

Significance Human breast cancer develops after a long period of latency under premalignant conditions. Strategies to target the premalignant conditions have yet to materialize since the molecular mechanisms remain obscure. Here, we discovered that FRS2β, expressed in a subset of mammary epithelial cells, directly activates nuclear factor–κB (NF-κB) and drives the initiation and promotion of the stroma-rich premalignant conditions. The FRS2β-triggered activation of NF-κB takes place in the early endosomes, the organelles, which have not been believed to be a major place for NF-κB signaling. The endosome signaling should be a novel focus for targeting therapy for prevention of breast cancer. This work paves a new way to develop preventive strategies of breast tumor development., Although it is held that proinflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we demonstrate that FRS2β, an adaptor protein expressed in a small subset of epithelial cells, triggers the proinflammatory changes that induce stroma in premalignant mammary tissues and is responsible for the disease onset. FRS2β deficiency in mouse mammary tumor virus (MMTV)–ErbB2 mice markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2β-deficient premalignant tissues. We found that colocalization of FRS2β and the NEMO subunit of the IκB kinase complex in early endosomes led to activation of nuclear factor–κB (NF-κB), a master regulator of inflammation. Moreover, inhibition of the activities of the NF-κB–induced cytokines, CXC chemokine ligand 12 and insulin-like growth factor 1, abrogated tumorigenesis. Human breast cancer tissues that express higher levels of FRS2β contain more stroma. The elucidation of the FRS2β–NF-κB axis uncovers a molecular link between the proinflammatory changes and the disease onset.

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, β, 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.

Background: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. Objectives: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. Method: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10–8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. Results: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). Conclusions: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.

Hypoxia-induced retinal edema primarily induced by vascular lesion is seen in various conditions such as diabetic retinopathy (DR) and retinal vein occlusion (RVO). The edematous changes in these conditions occur mainly in intermediate and deep layers of retina as a result of disruption of the inner blood-retinal barrier (iBRB). However, the effect of direct and acute hypoxia on iBRB remains to be elucidated. To investigate direct and acute hypoxia-induced changes in retina, especially in astrocytes/Müller cells that are involved in the maintenance of retinal structure and function, we developed an adult mouse model of hypoxia-induced retinal edema by 24-h exposure in a 6% oxygen environment. Immunohistochemical staining of glial fibrillary acidic protein (GFAP) was enhanced mainly in the superficial layer of the hypoxic retina, corresponding to edematous change. Electron microscopic observation of the hypoxic retina showed vacuole formation in astrocyte/Müller cell foot processes around capillaries in the superficial layer, while no abnormal findings in the perivascular areas were found in intermediate and deep layers. Increase in vascular leakage quantified by Evans blue dye and tight junction breakdown detected by electron-dense tracer were observed in the hypoxia group. In the hypoxic retina, microglia was activated and relative gene expressions of pro-inflammatory cytokines were significantly upregulated. Dexamethasone suppressed these hypoxia-induced pathological reactions. Thus, unlike DR and RVO that induce iBRB breakdown in deeper retinal layers, atmospheric hypoxia induced iBRB disruption with subsequent edematous change mainly in the superficial layer of the retina, and that dexamethasone prevented these pathological changes. In this mouse model, direct and acute hypoxia induces retinal edema in the superficial layer of the retina with morphological changes of astrocytes/Müller cells, and is potentially useful for ophthalmic research in the field related to retinal hypoxia and its treatment.

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.

Diabetic retinopathy is a major cause of blindness in developed countries, and is characterized by deterioration of barrier function causing vascular hyperpermeability and retinal edema. Vascular endothelial growth factor (VEGF) is a major mediator of diabetic macular edema. Although anti-VEGF drugs are the first-line treatment for diabetic macular edema, some cases are refractory to anti-VEGF therapy. Osteopontin (OPN) is a phosphoglycoprotein with diverse functions and expressed in various cells and tissues. Elevated OPN level has been implicated in diabetic retinopathy, but whether OPN is involved in hyperpermeability remains unclear. Using streptozotocin-induced diabetic mice (STZ mice) and human retinal endothelial cells (HRECs), we tested the hypothesis that up-regulated OPN causes tight junction disruption, leading to vascular hyperpermeability. The serum and retinal OPN concentrations were elevated in STZ mice compared to controls. Intravitreal injection of anti-OPN neutralizing antibody (anti-OPN Ab) suppressed vascular hyperpermeability and prevented decreases in claudin-5 and ZO-1 gene expression levels in the retina of STZ mice. Immunohistochemical staining of retinal vessels in STZ mice revealed claudin-5 immunoreactivity with punctate distribution and attenuated ZO-1 immunoreactivity, and these changes were prevented by anti-OPN Ab. Intravitreal injection of anti-OPN Ab did not change VEGF gene expression or protein concentration in retina of STZ mice. In an in vitro study, HRECs were exposed to normal glucose or high glucose with or without OPN for 48 h, and barrier function was evaluated by transendothelial electrical resistance and Evans blue permeation. Barrier function deteriorated under high glucose condition, and was further exacerbated by the addition of OPN. Immunofluorescence localization of claudin-5 and ZO-1 demonstrated punctate appearance with discontinuous junction in HRECs exposed to high glucose and OPN. There were no changes in VEGF and VEGF receptor-2 expression levels in HRECs by exposure to OPN. Our results suggest that OPN induces tight junction disruption and vascular hyperpermeability under diabetic conditions. Targeting OPN may be an effective approach to manage diabetic retinopathy.

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.