Diagnostic accuracy of fine-needle aspiration cytology of the breast in Japan: report from the Working Group on the Accuracy of Breast Fine-Needle Aspiration Cytology of the Japanese Society of Clinical Cytology.

The Working Group of the Japanese Society of Clinical Cytology was assembled to assess the current status of breast cytology in Japan by conducting a large-scale survey regarding the accuracy of fine-needle aspiration biopsy (FNAB) in Japan. We collected data and investigated the status of breast cytological diagnosis at 12 different cooperating facilities in Japan, and re-evaluated their false-negative and false-positive cases. Among 30,535 individuals who underwent a breast cytological examination, analyses were conducted on 10,890 individuals (35.7%) in whom cytological diagnoses were confirmed by histology. Among these patients, the cytological diagnosis had an inadequate rate of 17.7%, an indeterminate rate of 7.8%, a positive predictive value of 'malignancy suspected' cells of 92.4%, an absolute sensitivity of 76.7%, a complete sensitivity of 96.7%, a specificity of 84.3%, a positive predictive value of 'malignant' cells of 99.5%, a false-negative value of 3.31%, a false-positive value of 0.25% and an accuracy rate of 88.0%. Subsequently, 297 false-negative and 23 false-positive cases were re-evaluated and several factors were characterized (i.e. histological type, tumor size and misread points). This survey collected data from a large number of cases for breast FNAB. Based on our survey, the accuracy of FNAB in Japan was relatively high compared with the goal of assessment of diagnostic accuracy. However, there were some false-negative and false-positive cases. Improvements in accuracy resulting from the learning points in the present study will lead to more useful and reliable diagnostic tools in clinical practice.

Reactive oxygen species assay-based risk assessment of drug-induced phototoxicity: classification criteria and application to drug candidates.

We have previously demonstrated that the phototoxic potential of chemicals could be partly predicted by the determination of reactive oxygen species (ROS) from photo-irradiated compounds. In this study, ROS assay strategy was applied to 39 marketed drugs and 210 drug candidates in order to establish provisional classification criteria for risk assessment of drug-induced phototoxicity. The photosensitizing properties of 39 model compounds consisting of phototoxic and non-phototoxic chemicals, as well as ca. 210 drug candidates including 11 chemical series were evaluated using ROS assay and the 3T3 neutral red uptake phototoxicity test (NRU PT). With respect to marketed drugs, most phototoxic drugs tended to cause type I and/or II photochemical reactions, resulting in generation of singlet oxygen and superoxide. There seemed to be a clear difference between phototoxic drugs and non-phototoxic compounds in their abilities to induce photochemical reactions. A plot analysis of ROS data on the marked drugs provided classification criteria to discriminate the photosensitizers from non-phototoxic substances. Of all drug candidates tested, 35.2% compounds were identified as phototoxic or likely phototoxic on the basis of the 3T3 NRU PT, and all ROS data for these phototoxic compounds were found to be over the threshold value. Furthermore, 46.3% of non-phototoxic drug candidates were found to be in the subthreshold region. These results verify the usefulness of the ROS assay for understanding the phototoxicity risk of pharmaceutical substances, and the ROS assay can be used for screening purposes in the drug discovery stage.

In vitro phototoxic potential and photochemical properties of imidazopyridine derivative: a novel 5-HT4 partial agonist.

Drug-induced phototoxic skin responses have been recognized as undesirable side effects, and as we previously proposed the determination of reactive oxygen species (ROS) from photo-irradiated compounds can be effective for the prediction of phototoxic potential. In this investigation, we evaluated the photosensitizing properties of imidazopyridine derivative, a novel 5-HT(4) partial agonist, using ROS assay and several analytical/biochemical techniques. Exposure of the compound to simulated sunlight resulted in the significant production of singlet oxygen, which is indicative of its phototoxic potential. In practice, an imidazopyridine derivative under UVA/B light exposure also showed significant photodegradation and even photobiochemical events; peroxidation of fatty acid and genetic damage after DNA-binding, which are considered as causative agents for phototoxic dermatitis. Interestingly, both photodegradation and lipoperoxidation were dramatically attenuated by the addition of radical scavengers, especially singlet oxygen quenchers, suggesting the possible involvement of ROS generation in the phototoxic pathways. In the 3T3 neutral red uptake phototoxicity test, imidazopyridine derivative also showed the phototoxic effect on 3T3 mouse fibroblast cells. These results suggest the phototoxic risk of newly synthesized imidazopyridine derivative and also verify the usefulness of ROS assay for phototoxicity prediction.

Evaluation of hydrate formation of a pharmaceutical solid by using diffuse reflectance infrared Fourier-transform spectroscopy.

Ampicilline and nitrofurantoin, in both anhydrous and hydrate forms, were characterized by powder X-ray diffractometry (PXRD), thermogravimetric and differential thermal analyses (TG/DTA) and diffuse reflectance FT-IR spectroscopy (DRIFTS). Of all the analytical tools applied, only DRIFTS was able to indicate the formation of hydrogen bonds between the molecules of the anhydrous drug substance and crystalline water uptaken from atmospheric moisture as evidenced by the significant absorption at 3500-3700cm(-1) corresponding to crystal water. These results suggested that DRIFTS could provide information on hydration without a standard sample and accurately evaluate the physical stability focusing on the qualification of slight hydration in the early stages of pharmaceutical development. In addition, DRIFTS was applied to the besylate salt of pharmaceutical compound A to identify any possible hydration. This salt had the stable form BSA-I, metastable form BSA-II and hydrate form BSA-III. DRIFTS was able to show the hydration of BSA-II even when stored in a capped bottle, eventually leading to the transformation into BSA-III, which was not detected by PXRD. These findings verify the usefulness of DRIFTS for the solid-state characterization of pharmaceutical substances, especially the monitoring of gradual hydration.

In vitro phototoxicity of dihydropyridine derivatives: a photochemical and photobiological study.

Some photosensitizing drugs can cause phototoxic skin responses even after systemic administration; therefore, avoidance of undesired side-effects is a key consideration in drug discovery and development. As a prediction tool for phototoxic risk, we previously proposed the monitoring of reactive oxygen species (ROS) generated from compounds irradiated with UVA/B, which can be effective for understanding photochemical/photobiological properties. In this investigation, we evaluated the photosensitizing properties of a novel dihydropyridine derivative, with bradykinin B(2) receptor antagonist activity (compound A) using our ROS assay and several analytical/biochemical techniques. Exposure of compound A, and several dihydropyridine-type calcium channel antagonists to simulated sunlight resulted in the significant production of singlet oxygen, superoxide, or both, which indicates their photosensitive/phototoxic potential. This is consistent with the observation that compound A under UVA/B light exposure caused significant photodegradation and even peroxidation of fatty acid, which could lead to phototoxic dermatitis. Interestingly, the addition of radical scavengers, especially GSH, MPG and BHA, could attenuate the lipid peroxidation, suggesting the involvement of ROS generation in the phototoxic pathways of compound A. In the 3T3 neutral red uptake phototoxicity test, compound A also showed a phototoxic effect on 3T3 mouse fibroblast cells. These findings also support the usefulness of the ROS assay for the risk assessment studies on the drug-induced phototoxicity even at the early stages of pharmaceutical development.

Fundamental studies on electrokinetic chromatography with PEGylated phospholipid micelles.

In this study, micelles prepared from distearoylphosphatidylethanolamine with covalently attached poly(ethylene) glycol) (PEG) of molecular weight 2000 (DSPE-PEG-2000) were employed in micellar electrokinetic chromatography (MEKC) as pseudostationary phases. Since DSPE-PEG-2000 contains long hydrophobic alkyl chains, an anionic phosphate group, and hydrophilic PEG chains, the prepared micelles are expected to provide a characteristic retention behavior for both neutral and ionic compounds. As a typical example, a baseline separation of phenol and 2-naphthol was successfully achieved by using the DSPE-PEG-2000 micelles as a background electrolyte for MEKC; such success clearly shows that the micelles can retain electrically neutral compounds. The MEKC separations of anionic and cationic compounds with a DSPE-PEG-2000 micellar solution and the enantioseparation of binaphthyl compounds with mixed micelles containing bile salt are also discussed.

High-throughput reactive oxygen species (ROS) assay: an enabling technology for screening the phototoxic potential of pharmaceutical substances.

Recently, attention has been drawn to drug-induced phototoxic skin responses, and avoidance of this undesired side effect is necessary for pharmaceutical development. We previously proposed that determination of reactive oxygen species (ROS) generated from photoirradiated compounds would be effective for the prediction of the phototoxic potential. In this investigation, a high-throughput ROS assay system was developed using a multiwell plate and quartz reaction container. The experimental conditions of irradiance uniformity, UV intensity, exposure time, temperature and solvent systems were found to affect the generation of ROS, and thus the conditions of the ROS assay were optimized. The intra- and inter-day R.S.D. values for the determination of ROS from quinine (200 microM) irradiated at 250 W/m(2) for 1h was found to be less than 3.3 and 4.5%, respectively. The results from the ROS assay of 39 compounds allowed us to estimate classification criteria to identify the ability of phototoxic/photochemical responses. The developed assay system will be an effective tool for predicting the phototoxic potential of pharmaceutical candidates in early stage of pharmaceutical development.

Analytical studies on photochemical behavior of phototoxic substances; effect of detergent additives on singlet oxygen generation.

PURPOSE: Monitoring of reactive oxygen species (ROS) generation from photoirradiated compounds would be effective for the prediction of the phototoxic potential. The aim of this investigation was to clarify the possible role of biomimetic vehicle systems on the photochemical properties of phototoxic compounds, focusing on the singlet oxygen generation. MATERIALS AND METHODS: Nine phototoxic and one non-phototoxic compounds (200 microM), dissolved in Tween 20, sodium laurate, or sodium dodecyl sulfate (SDS) micellar solution, were exposed to UVA/B light (250 W/m2), and singlet oxygen generation was monitored by RNO bleaching methodology. Photochemical properties of photosensitizers were also evaluated by UV measurement, and the interaction of photosensitizers with surfactant micelles was assessed by Z-potential and NMR spectroscopic analyses. RESULTS: All phototoxic compounds tended to generate singlet oxygen under light exposure in the all micellar solutions tested. There appeared to be some differences in photoreactivity of both cationic and anionic photosensitizers among the micelles tested, whereas ROS data on anthracene, dissolved in three micellar solutions, were found to be quite similar. Photosensitizers exhibited no significant changes in UV spectral patterns among the dissolving micellar solutions. Addition of cationic photosensitizer at the final concentration of 100 microM into 100 mM SDS solution resulted in the 20 mV increase of zeta potential and transition of NMR spectral pattern, which would reflect the electrostatic interaction with anionic micelles. CONCLUSION: Based on the data obtained, the photoreactivity of photosensitizing molecules, especially cationic and anionic photosensitizers, strongly depends on the physicochemical properties of the microenvironment.

Capillary electrophoretic studies on the photogenotoxic potential of pharmaceutical substances.

Drug-induced phototoxic skin responses, including photoirritation, photoallergy and photogenotoxicity, are identified as adverse reactions. In this study, we attempted to develop effective analytical tools to predict the photogenotoxic potential of pharmaceutical substances with the use of pBR322 DNA, a plasmid DNA. pBR322 DNA was irradiated with simulated solar light in the presence of photosensitizers, and its structural conversion was assessed by agarose gel electrophoresis (AGE), transmission electron microscopy (TEM) and capillary gel electrophoresis (CGE). The generation of reactive oxygen species (ROS) from photoirradiated photosensitizers was also assessed by spectrophotometrical determination. Concomitant ultraviolet (UV) exposure of pBR322 DNA and photosensitizers resulted in significant oxidative damage to the DNA, as evidenced by AGE, TEM and CGE data, indicating a structural transition from supercoiled form to open circular form. Photosensitizer-induced DNA damage was attenuated by the addition of radical scavengers, especially sodium azide, a typical scavenger of singlet oxygen (1 O2), and enhanced by the addition of deuterium water, an enhancer of the life time of 1 O2. These data, taken together with the results of the ROS assay, suggest that singlet oxygen might act as a major toxic species in quinine-induced photogenotoxicity. The structural analysis of plasmid DNA by CGE after exposure to UVA/B in the presence of photosensitizers could be automated, allowing easy, fast and highly reliable prediction for the photogenotoxic potential of a large number of drug candidates.

Photoreactivity of amino acids: tryptophan-induced photochemical events via reactive oxygen species generation.

Tryptophan (Trp), an aromatic amino acid, is a constituent of peptides/proteins and is also a precursor of serotonin, kynurenine derivatives, and nicotinamide adenine dinucleotides. There have been a number of reports on photochemical reactions involving peptides/proteins which contain Trp that showed significant photodegradation, dimerization, and photoionization. The photochemical properties of Trp have not been fully elucidated, and this would provide novel insight into the handling of Trp-containing peptides/proteins. Consequently, we have been trying to evaluate the photochemical properties of Trp, as well as other essential amino acids, focusing on their photosensitivity, photodegradation, and their ability to induce lipid peroxidation. Among all the essential amino acids tested, Trp exhibited the maximal level of superoxide anion generation under 18 h of light exposure (30000 lux). UV spectral analysis of Trp suggested the absorbability of UVA/B light, and exposure of Trp, in both solid and solution states, to UVA/B light resulted in significant photodegradation (t(0.5): 18 h) and gradual color changes. In addition, photoirradiated Trp generated lipoperoxidant, a causative agent of photoirritation, and this might be associated with ROS generation.

The role of degradant profiling in active pharmaceutical ingredients and drug products.

Forced degradation studies are used to facilitate the development of analytical methodology, to gain a better understanding of active pharmaceutical ingredient (API) and drug product (DP) stability, and to provide information about degradation pathways and degradation products. In order to fulfill development and regulatory needs, this publication provides a roadmap for when and how to perform studies, helpful tools in designing rugged scientific studies, and guidance on how to record and communicate results.

Electrophoretic analysis of proteins and enantiomers using capillaries modified by a successive multiple ionic-polymer layer (SMIL) coating technique.

The applicability of three-layer coatings consisting of three different polymers (A(+)-B(-)-C(+) coating) prepared by a successive multiple ionic-polymer layer (SMIL) coating technique to the immobilization of polypeptides and/or proteins onto the inner surface of the capillaries was investigated to provide a high-performance separation medium for proteins and enantiomers in capillary electrophoresis (CE). To obtain a stable protein-coated capillary, high molecular mass poly(ethyleneimine) (PEI) was employed as the first layer in the A(+)-B(-)-C(+) coating, and then a cationic protein was immobilized as the third layer. Comparisons of analytical performances between the A(+)-B(-)-C(+) coating and the conventional SMIL-coated (A(+)-B(-)-A(+) coating) capillary were conducted. The CE separation of cationic proteins was successfully achieved with the prepared capillaries. In addition, the polypeptide- and protein-coated capillaries were applied to the chiral separation of a binaphthyl compound. It should be noted that the chiral separation efficiency was strongly dependent on the second anionic polymer layer of the coating. Effects of the interaction between oppositely charged ionic polymer layers on the separation efficiency are discussed.

Effect of 2-methoxyestradiol, buthionine sulfoximine and hydrogen peroxide on the viability of renal carcinoma cell lines (ACHN and ACVB).

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17beta-estradiol, induces the intracellular accumulation of superoxide anion (O2*-) and buthionine sulfoximine (BSO) is an inhibitor of glutathione (GSH) synthesis. We have examined the combination anticancer effect of 2-ME and BSO accompanied with hydrogen peroxide (H2O2). 2-ME inhibited cell growth in renal carcinoma cell lines (ACHN and ACVB) accompanied by an increase in the intracellular contents of GSH. The combination of 2-ME, BSO and H2O2 showed a significant antiproliferation effect in both ACHN and ACVB. The intracellular levels of reactive oxygen species (ROS) with a combination with 2-ME and H2O2 in ACHN and ACVB pretreated with BSO were markedly increased, which may have contributed to the potential antiproliferative action.

Analytical studies on the prediction of photosensitive/phototoxic potential of pharmaceutical substances.

PURPOSE: Phototoxic responses after administration of photosensitive pharmaceutics have been recognized as undesirable side effects, and predicting potential hazardous side effects is gaining importance as new drugs are introduced to the market. In this work, we characterize the photochemical/photobiological properties of model compounds to develop an effective screening method for the prediction of phototoxic/photosensitive potential. METHODS: Twenty-one known photosensitive/phototoxic compounds and five weak/nonphototoxic compounds were subjected to ultraviolet (UV) spectral analyses and photochemical evaluation including the determination of produced reactive oxygen species (ROS) and photostability study. The photooxidation of linoleic acid was also monitored in the presence of tested compounds, guided on the formation of thiobarbituric acid reactive substances. RESULTS: Most photosensitive/phototoxic drugs tested, even weak UV absorbers, at a concentration of 200 microM showed significant production of ROS under 18 h light exposure (30,000 lx). On the other hand, ROS generated from weak/nonphototoxic compounds, including strong UV absorber benzocaine, were low or negligible. Although exposure of quinine to light resulted in significant degradation (half-life, t1/2=6.4 h), it was dramatically attenuated by the addition of ROS scavengers, especially sodium azide (t1/2=122.6 h). Furthermore, concomitant exposure of photosensitive/phototoxic compounds (200 microM) and linoleic acid (1 mM) for 18 h led to the marked formation of lipoperoxide. CONCLUSION: Results indicated that known photosensitive/phototoxic compounds tested have the ability to generate ROS under light exposure, and this photochemical reaction could be associated with their photoinstability and/or phototoxic responses. Based on these findings, determination of ROS, generated from photoirradiated compounds, may be an effective predictive model in recognizing their photosensitive/phototoxic potential.