Simultaneous determination of fourteen components of Gumiganghwal-tang tablet in human plasma by UPLC-ESI-MS/MS and its application to pharmacokinetic study

Gumiganghwal-tang is a traditional herbal medicine widely used for its anti-inflammatory, analgesic, and antipyretic effects. However, the safety and efficacy of its active ingredients based on an in vivo pharmacokinetic (PK) study have yet been investigated. We have established a sensitive and accurate UPLC-ESI-MS/MS method and conducted a PK study on 14 constituents of Gumiganghwal-tang through human plasma analysis. Analytical conditions were optimized according to the physicochemical properties of the 14 compounds to facilitate efficient separation and eliminate overlap or interference between peaks. KINETEX-C₁₈ and Inertsil-C₈ columns were used as UPLC stationary phases, and acetonitrile and aqueous formic acid were used as mobile phases. All the analytes were quantified with a triple quadrupole mass spectrometer using electrospray ionization in multiple reaction monitoring mode. The chromatograms of 14 bioactive compounds showed excellent elution and sensitivity, and each peak was selectively separated and quantified without interference with each other or impurities. The established analytical method was based on international guidelines and was successfully used to perform PK studies of 14 herbal ingredients in humans after oral administration with Gumiganghwal-tang tablets. The oral absorption of most active components of Gumiganghwal-tang was relatively rapid and remained considerably long in the body to be quantified in plasma up to 48 h after administration.     

Solid phase microextraction chemical biopsy tool for monitoring of doxorubicin residue during in vivo lung chemo-perfusion

Development of a novel in vivo lung perfusion (IVLP) procedure allows localized delivery of high-dose doxorubicin (DOX) for targeting residual micrometastatic disease in the lungs. However, DOX delivery via IVLP requires careful monitoring of drug level to ensure tissue concentrations of this agent remain in the therapeutic window. A small dimension nitinol wire coated with a sorbent of biocompatible morphology (Bio-SPME) has been clinically evaluated for in vivo lung tissue extraction and determination of DOX and its key metabolites. The in vivo Bio-SPME-IVLP experiments were performed on pig model over various (150 and 225 mg/m²) drug doses, and during human clinical trial. Two patients with metastatic osteosarcoma were treated with a single 5 and 7 μg/mL (respectively) dose of DOX during a 3-h IVLP. In both pig and human cases, DOX tissue levels presented similar trends during IVLP. Human lung tissue concentrations of drug ranged between 15 and 293 μg/g over the course of the IVLP procedure. In addition to DOX levels, Bio-SPME followed by liquid chromatography-mass spectrometry analysis generated 64 metabolic features during endogenous metabolite screening, providing information about lung status during drug administration. Real-time monitoring of DOX levels in the lungs can be performed effectively throughout the IVLP procedure by in vivo Bio-SPME chemical biopsy approach. Bio-SPME also extracted various endogenous molecules, thus providing a real-time snapshot of the physiology of the cells, which might assist in the tailoring of personalized treatment strategy.     

One extraction tool for in vitro-in vivo extrapolation? SPME-based metabolomics of in vitro 2D, 3D,and in vivo mouse melanoma models

Solid phase microextraction (SPME) in combination with high-resolution mass spectrometry was employed for the determination of metabolomic profile of mouse melanoma growth within in vitro 2D, in vitro 3D, and in vivo models. Such multi-model approach had never been investigated before. Due to the low-invasiveness of SPME, it was possible to perform time-course analysis, which allowed building time profile of biochemical reactions in the studied material. Such approach does not require the multiplication of samples as subsequent analyses are performed from the very same cell culture or from the same individual. SPME already reduces the number of animals required for experiment; therefore, it is with good concordance with the 3Rs rule (replacement, reduction, and refinement). Among tested models, the largest number of compounds was found within the in vitro 2D cell culture model, while in vivo and in vitro 3D models had the lowest number of detected compounds. These results may be connected with a higher metabolic rate, as well as lower integrity of the in vitro 2D model compared to the in vitro 3D model resulting in a lower number of compounds released into medium in the latter model. In terms of in vitro-in vivo extrapolation, the in vitro 2D model performed more similar to in vivo model compared to in vitro 3D model; however, it might have been due to the fact that only compounds secreted to medium were investigated. Thus, in further experiments to obtain full metabolome information, the intraspheroidal assessment or spheroid dissociation would be necessary.     

Effect of Dosage Form Properties on Therapeutic Efficacy of Tolbutamide Tablets

The rate of gastrointestinal absorption and the physiologic availability of tolbutamide are functions of the dissolution rate of the drug in gastrointestinal fluids. This property can be modified markedly by pharmaceutical formulation factors. The in vitro dissolution rates of clinically efficacious and clinically inefficacious tolbutamide tablets have been determined, and it has been found that the former dissolved considerably more rapidly than the latter. Once diabetic patients have been “titrated” with a particular brand of tolbutamide they should not be switched to a different brand without “retitration.” There is need to establish an adequate in vitro test to assure the physiologic availability of tolbutamide from commercial tablets.     

Synthesis and electrochemical properties of environmental free l-glutathione grafted graphene oxide/ZnO nanocomposite for highly selective piroxicam sensing

A simple and reliable strategy was proposed to engineer the glutathione grafted graphene oxide/ZnO nanocomposite (glutathione-GO/ZnO) as electrode material for the high-performance piroxicam sensor. The prepared glutathione-GO/ZnO nanocomposite was well characterized by X-ray diffraction (XRD), Fourier transform infrared spectrum (FTIR), X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FE-SEM), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The novel nanocomposite modified electrode showed the highest electrocatalytic activity towards piroxicam (oxidation potential is 0.52 V). Under controlled experimental parameters, the proposed sensor exhibited good linear responses to piroxicam concentrations ranging from 0.1 to 500 μM. The detection limit and sensitivity were calculated as 1.8 nM and 0.2 μA/μM·cm², respectively. Moreover, it offered excellent selectivity, reproducibility, and long-term stability and can effectively ignore the interfering candidates commonly existing in the pharmaceutical tablets and human fluids even at a higher concentration. Finally, the reported sensor was successfully employed to the direct determination of piroxicam in practical samples.     

Transformation of berberine to its demethylated metabolites by the CYP51 enzyme in the gut microbiota

Berberine (BBR) is an isoquinoline alkaloid extracted from Coptis chinensis that improves diabetes, hyperlipidemia and inflammation. Due to the low oral bioavailability of BBR, its mechanism of action is closely related to the gut microbiota. This study focused on the CYP51 enzyme of intestinal bacteria to elucidate a new mechanism of BBR transformation by demethylation in the gut microbiota through multiple analytical techniques. First, the docking of BBR and CYP51 was performed; then, the pharmacokinetics of BBR was determined in ICR mice in vivo, and the metabolism of BBR in the liver, kidney, gut microbiota and single bacterial strains was examined in vitro. Moreover, 16S rRNA analysis of ICR mouse feces indicated the relationship between BBR and the gut microbiota. Finally, recombinant E. coli containing cyp51 gene was constructed and the CYP51 enzyme lysate was induced to express. The metabolic characteristics of BBR were analyzed in the CYP51 enzyme lysate system. The results showed that CYP51 in the gut microbiota could bind stably with BBR, and the addition of voriconazole (a specific inhibitor of CYP51) slowed down the metabolism of BBR, which prevented the production of the demethylated metabolites thalifendine and berberrubine. This study demonstrated that CYP51 promoted the demethylation of BBR and enhanced its intestinal absorption, providing a new method for studying the metabolic transformation mechanism of isoquinoline alkaloids in vivo.     

瑞格列奈片的制备和质量标准研究

目的制备瑞格列奈片,并建立其质量标准。方法以溶出度、硬度、外观为指标,通过正交试验L9（34）筛选出最优处方;采用高效液相法测定瑞格列奈的含量、含量均匀度和溶出度。结果优选出的瑞格列奈片的处方组成为：微晶纤维素∶PVP-K30∶CCNa的质量比为74∶5∶20;含量测定方法学考察结果：瑞格列奈质量浓度在9.90599.05μg.mL^-1范围内与峰面积呈良好线性关系,回收率为100.83%、RSD为0.98%（n=9）,最低检测限为2.0 ng,最低定量限为8.0 ng;含量均匀度为6.84;30 min累积溶出度为98.25%。结论筛选的处方合理,制备工艺简单可行,制备的瑞格列奈片符合制剂质量要求。     

Rates of Dissolution of Tolbutamide Tablets

An investigation was made of the pharmaceutical properties and the in vitro dissolution rates of 18 commercially available brands of tolbutamide tablets, all of which met the limits set by the Food and Drug Regulations for tablets sold in Canada.

A marked variation in dissolution rates was found, which bore no relation to the official disintegration time. These wide variations in dissolution rate point to a need for (a) a comprehensive study of the in vivo effects of different tolbutamide tablets, and (b) an official test that sets limits for the rate of dissolution of tolbutamide tablets, in addition to the one that places limits on disintegration time.

     

Neuroprotective effects of Ginkgo biloba dropping pills in Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease in the world; however, it lacks effective and safe treatments. Ginkgo biloba dropping pill (GBDP), a unique Chinese G. biloba leaf extract preparation, exhibits antioxidant and neuroprotective effects and has a potential as an alternative therapy for PD. Thus, the aims of this study were to evaluate the effects of GBDP in in vitro and in vivo PD models and to compare the chemical constituents and pharmacological activities of GBDP and the G. biloba extract EGb 761. Using liquid chromatography tandem-mass spectrometry, 46 GBDP constituents were identified. Principal component analysis identified differences in the chemical profiles of GBDP and EGb 761. A quantitative analysis of 12 constituents showed that GBDP had higher levels of several flavonoids and terpene trilactones than EGb 761, whereas EGb 761 had higher levels of organic acids. Moreover, we found that GBDP prevented 6-hydroxydopamine-induced dopaminergic neuron loss in zebrafish and improved cognitive impairment and neuronal damage in methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD mice. Although similar effects were observed after EGb 761 treatment, the neuroprotective effects were greater after GBDP treatment on several endpoints. In addition, in vitro results suggested that the Akt/GSK3β pathway may be involved in the neuroprotective effects of GBDP. These findings demonstrated that GBDP have potential neuroprotective effects in the treatment of PD.     

Development of a rapid GC-FID method to simultaneously determine triethylamine,diisopropylamine, and 1,1,3,3-tetramethylguanidine residues in an active pharmaceutical ingredient

A rapid GC-FID method was developed to simultaneously determine residual levels of triethylamine (TEA), 1,1,3,3-tetramethylguanidine (TMG), and diisopropylamine (DIPA) in the synthetic route of an active pharmaceutical ingredient (API). Due to the severe absorption of amines on GC stationary phases, GC columns with various stationary phases were evaluated for optimal peak shape and reproducibility. The final conditions used the Agilent CP-Volamine column to resolve the three amines in 12 min. Various inlet liners were also screened to further improve the sensitivity of the analysis. The Restek Siltek® liner was selected to achieve the desired detectability for the method. The quantitation limits were 4, 3, and 4 μg/mL for TEA, DIPA, and TMG in the presence of API, respectively. All three amines showed good linearity (r > 0.999) and recoveries (> 90%) over the concentration range of 3 to 16 μg/mL. The testing of residual amines was initially performed at the penultimate stage of the synthesis. However, this work demonstrates that TMG can act as a proton sponge to react with salicylic acid, the counter ion of the penultimate, to form a volatile component that elutes at a different retention time. Consequently, in the final method, these three amines were monitored in the final API to circumvent the matrix interference. Key parameters of the method were qualified per method validation requirements in ICH guidelines. The method was successfully applied for batch testing during development and implemented as an in-process control procedure at manufacturing sites.     

In vitro fertilisation in domestic mammals—a brief overview

Many factors influence the final oocyte maturation, fertilisation, and early embryo development, and there are both similarities and differences between species. When comparing the advancement of assisted reproductive technologies (ARTs), the development in the bovine species is not far behind the medical front, with around one million in vitro-produced bovine embryos each year. This rate of progress is not seen in the other domestic species. This review aims to give an overview of the development and specific difficulties of in vitro embryo production in various domestic animal species, with the main focus on cows, pigs, and cats. In production animals, the aim of ARTs is commonly to increase the genetic progress, not to treat reproductive failure. The ARTs are also used for preservation of genetic diversity for the future. However, specifically for oocyte maturation, fertilisation, and early embryonic development, domestic mammals such as the cow and pig can be used as models for humans. This is particularly attractive from an animal welfare point of view since bovine and porcine oocytes are available in large numbers from discarded slaughterhouse material, thereby decreasing the need for research animals. Both for researchers on the animal and human medical fronts, we aim for the development of in vitro production systems that will produce embryos and offspring that are no different from those conceived and developed in vivo. Species-comparative research and development can provide us with crucial knowledge to achieve this aim and hopefully help us avoid unnecessary problems in the future.     

Taxifolin stability: In silico prediction and in vitro degradation with HPLC-UV/UPLC–ESI-MS monitoring

Taxifolin has a plethora of therapeutic activities and is currently isolated from the stem bark of the tree Larix gmelinni (Dahurian larch). It is a flavonoid of high commercial interest for its use in supplements or in antioxidant-rich functional foods. However, its poor stability and low bioavailability hinder the use of flavonoid in nutritional or pharmaceutical formulations. In this work, taxifolin isolated from the seeds of Mimusops balata, was evaluated by in silico stability prediction studies and in vitro forced degradation studies (acid and alkaline hydrolysis, oxidation, visible/UV radiation, dry/humid heating) monitored by high performance liquid chromatography with ultraviolet detection (HPLC-UV) and ultrahigh performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). The in silico stability prediction studies indicated the most susceptible regions in the molecule to nucleophilic and electrophilic attacks, as well as the sites susceptible to oxidation. The in vitro forced degradation tests were in agreement with the in silico stability prediction, indicating that taxifolin is extremely unstable (class 1) under alkaline hydrolysis. In addition, taxifolin thermal degradation was increased by humidity. On the other hand, with respect to photosensitivity, taxifolin can be classified as class 4 (stable). Moreover, the alkaline degradation products were characterized by UPLC-ESI-MS/MS as dimers of taxifolin. These results enabled an understanding of the intrinsic lability of taxifolin, contributing to the development of stability-indicating methods, and of appropriate drug release systems, with the aims of preserving its stability and improving its bioavailability.     

Comprehensive metabolic profiling of Alismatis Rhizoma triterpenes in rats based on characteristic ions and a triterpene database

Alismatis Rhizoma (AR) is widely used in Chinese medicine, and its major bioactive components, triterpenes, reportedly possess various pharmacological activities. Therefore, it is very important to study the metabolism of triterpenes in vivo. However, the metabolism of AR triterpene extract has not been comprehensively elucidated due to its complex chemical components and metabolic pathways. In this study, an ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method, which was based on the characteristic ions from an established database of known triterpenes, was used to analyze the major metabolites in rats following the oral administration of Alismatis Rhizoma extracts (ARE). As a result, a total of 233 constituents, with 85 prototype compounds and 148 metabolites, were identified for the first time. Hydrogenation, oxidation, sulfate and glucuronidation conjugation were the major metabolic pathways for triterpenes in AR. In addition, the mutual in vivo transformation of known ARE triterpenes was discovered and confirmed for the first time. Those results provide comprehensive insights into the metabolism of AR in vivo, which will be useful for future studies on its pharmacodynamics and pharmacokinetics. Moreover, this established strategy may be useful in metabolic studies of similar compounds.     

Simultaneous enantioseparation and simulation studies of atenolol,metoprolol and propranolol on Chiralpak® IG column using supercritical fluid chromatography

Enantioseparation of three β-blockers, i.e., atenolol, metoprolol and propranolol, was studied on amylose tris(3-chloro-5-methylphenylcarbamate) immobilized chiral stationary phase using supercritical fluid chromatography (SFC). The effect of organic modifiers (methanol, isopropanol and their mixture), column temperature and back pressure on chiral separation of β-blockers was evaluated. Optimum chromatographic separation with respect to resolution, retention, and analysis time was achieved using a mixture of CO₂ and 0.1% isopropyl amine in isopropanol: methanol (50:50, V/V), in 75:25 (V/V) ratio. Under the optimized conditions, the resolution factors (R_s) and separation factors (α) were greater than 3.0 and 1.5, respectively. Further, with increase in temperature (25–45 °C) and pressure (100–150 bars) there was corresponding decrease in retention factors (k), α and R_s. However, a reverse trend (α and R_s) was observed for atenolol with increase in temperature. The thermodynamic data from van''t Hoff plots revealed that the enantioseparation was enthalpy driven for metoprolol and propranolol while entropy driven for atenolol. To understand the mechanism of chiral recognition and the elution behavior of the enantiomers, molecular docking studies were performed. The binding energies obtained from simulation studies were in good agreement with the elution order found experimentally and also with the free energy values. The method was validated in the concentration range of 0.5–10 μg/mL for all the enantiomers. The limit of detection and limit of quantitation ranged from 0.126 to 0.137 μg/mL and 0.376–0.414 μg/mL, respectively. The method was used successfully to analyze these drugs in pharmaceutical preparations.     

血液细菌培养及药敏分析

目的：了解血液感染菌及其耐药性,以便指导临床合理用药.方法：采用BACT/ALERT 3D型全自动血培养仪对收集的3 482份血标本进行培养,培养出病原菌的阳性标本用ATB-EXPRESS型全自动细菌鉴定仪进行鉴定,用K-B纸片法进行体外药敏试验.结果：血培养细菌阳性率7.2％;表皮葡萄球菌、大肠埃希菌、溶血葡萄球菌、金黄色葡萄球菌、嗜麦芽窄食单胞菌为前5位优势菌;G＋球菌优势菌对万古霉素、米诺环素耐药率少于7.0％,G-菌优势菌对阿米卡星、哌拉西林/他唑巴坦、阿莫西林/克拉维酸耐药率少于10.0％.结论：血液感染病原菌种类比较多,耐药差异比较大.     

Design and preparation of a new multi-targeted drug delivery system using multifunctional nanoparticles for co-delivery of siRNA and paclitaxel

Drug resistance is a great challenge in cancer therapy using chemotherapeutic agents. Administration of these drugs with siRNA is an efficacious strategy in this battle. Here, the present study tried to incorporate siRNA and paclitaxel (PTX) simultaneously into a novel nanocarrier. The selectivity of carrier to target cancer tissues was optimized through conjugation of folic acid (FA) and glucose (Glu) onto its surface. The structure of nanocarrier was formed from ternary magnetic copolymers based on FeCo-polyethyleneimine (FeCo-PEI) nanoparticles and polylactic acid-polyethylene glycol (PLA-PEG) gene delivery system. Biocompatibility of FeCo-PEI-PLA-PEG-FA(NPsA), FeCo-PEI-PLA-PEG-Glu (NPsB) and FeCo-PEI-PLA-PEG-FA/Glu (NPsAB) nanoparticles and also influence of PTX-loaded nanoparticles on in vitro cytotoxicity were examined using MTT assay. Besides, siRNA-FAM internalization was investigated by fluorescence microscopy. The results showed the blank nanoparticles were significantly less cytotoxic at various concentrations. Meanwhile, siRNA-FAM/PTX encapsulated nanoparticles exhibited significant anticancer activity against MCF-7 and BT-474 cell lines. NPsAB/siRNA/PTX nanoparticles showed greater effects on MCF-7 and BT-474 cells viability than NPsA/siRNA/PTX and NPsB/siRNA/PTX. Also, they induced significantly higher anticancer effects on cancer cells compared with NPsA/siRNA/PTX and NPsB/siRNA/PTX due to their multi-targeted properties using FA and Glu. We concluded that NPsAB nanoparticles have a great potential for co-delivery of both drugs and genes for use in gene therapy and chemotherapy.     

Determination of inhibitory activity of Salvia miltiorrhiza extracts on xanthine oxidase with a paper-based analytical device

A novel paper-based analytical device (PAD) was prepared and applied to determine the xanthine oxidase (XOD) inhibitory activity of Salvia miltiorrhiza extracts (SME). First, polycaprolactone was 3D printed on filter paper and heated to form hydrophobic barriers. Then the modified paper was cut according to the specific design. Necessary reagents including XOD for the colorimetric assay were immobilized on two separate pieces of paper. By simply adding phosphate buffer, the reaction was performed on the double-layer PAD. Quantitative results were obtained by analyzing the color intensity with the specialized device system (consisting of a smartphone, a detection box and sandwich plates). The 3D-printed detection box was small, with a size of 9.0 cm × 7.0 cm × 11.5 cm. Color component G performed well in terms of linearity and detection limits and thus was identified as the index. The reaction conditions were optimized using a definitive screening design. Moreover, a 10% glycerol solution was found to be a suitable stabilizer. When the stabilizer was added, the activity of XOD could be maintained for at least 15 days under 4 °C or −20 °C storage conditions. The inhibitory activity of SME was investigated and compared to that of allopurinol. The results obtained with the PAD showed agreement with those obtained with the microplate method. In conclusion, the proposed PAD method is simple, accurate and has a potential for point-of-care testing. It also holds promise for use in rapid quality testing of medicinal herbs, intermediate products, and preparations of traditional Chinese medicines.     

熊去氧胆酸片溶出度差异分析

目的比较国内不同厂家熊去氧胆酸片溶出度差异,以便对其质量进行更好地控制。方法分别考察了2种不同溶出介质和不同转速对溶出曲线的影响,并以磷酸盐缓冲液（pH7.5）为溶出介质,转速50r/min进行HPLC分析,计算不同时间点熊去氧胆酸的溶出率,利用相似度值（AV值）对溶出曲线进行相似度比较,并对溶出均匀度（A）进行评价。结果国内14家生产企业中有5家的产品溶出曲线与参比溶出曲线相似,仅有3家企业的所有产品溶出均匀性较好。结论本研究建立的方法能有效对熊去氧胆酸片的工艺水平和质量进行考察,可作为该品种质量控制的重要补充。     

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer. Eupatorin, a flavonoid with a wide range of phytomedical activities, suppresses proliferation of and induces apoptosis of multiple cancer cell lines. However, low solubility, poor bioavailability, and rapid degradation limit its efficacy. The aim of our study was to evaluate whether the use of mPEG-b-poly (lactic-co-glycolic) acid (PLGA) coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines. Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology, surface charge, particle size, drug loading, encapsulation efficiency and in vitro drug-release profile. The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test. Apoptosis was then determined by Hoechest staining, cell cycle analysis, NO production, annexin/propidium iodide (PI) assay, and Western blotting. The results indicated that eupatorin was successfully entrapped in Fe₃O₄@mPEG-b-PLGA nanoparticles with an efficacy of (90.99 ± 2.1)%. The nanoparticle’s size was around (58.5 ± 4) nm with a negative surface charge [(−34.16 ± 1.3) mV]. In vitro release investigation showed a 30% initial burst release of eupatorin in 24 h, followed by sustained release over 200 h. The MTT assay indicated that eupatorin-loaded Fe₃O₄@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 μM and 75 μM, respectively. Next, apoptosis was confirmed by nuclear condensation, enhancement of cell population in the sub-G1 phase and increased NO level. Annexin/PI analysis demonstrated that eupatorin-loaded Fe₃O₄@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency. Finally, Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles. Encapsulation of eupatorin in Fe₃O₄@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin. Based on these results, this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose, making it a suitable candidate for pharmacological uses.