Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution

Purpose

Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column.

Methods

Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8 kDa to 150kDa were assessed with and without cyclodextrin.

Results

Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied.

Discussion

High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products.

Conclusion

Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.

     

Neuropsychiatric clinical manifestations in elderly patients treated with hydroxychloroquine: a review article

Little is known about the development of psychosis during hydroxychloroquine (HCQ) treatment, especially in elderly patients affected by rheumatic diseases, with multiple comorbidities and treatments. To summarize the available evidence on HCQ-induced psychosis in elders, we performed a literature review. Additionally, individual case safety reports sent to the European Pharmacovigilance database (EudraVigilance) with HCQ as suspected drug and related to adverse events belonging to the System Organ Class ‘Psychiatric disorders’ were shown. Over the years, evidence was published about the risk of neuropsychiatric clinical manifestations during HCQ treatment for rheumatic diseases, but few of them were related to elderly patients. These adverse events can include less severe clinical manifestations such as affect lability and nervousness or more severe conditions such as actual psychosis and suicidal tendencies, which frequency are actually unknown. The presence of risk factors in these patients may precipitate HCQ-induced psychosis and their precocious detection could be associated with a risk minimization. Among predisposing risk factors, there are the co-exposure to interacting drugs, alcohol intake, familial history of psychiatric diseases, female gender, and the concomitant use of low-dose glucocorticoids. In some cases it was possible to reverse psychotic behaviour with the antipsychotic treatment or with HCQ suspension.     

Grape polyphenols corrects ageing-related detriments in neutrophil functionality via modulation of specific molecular targets

Oxidative stress and inflammation are intricately interlinked as aetiological factors in the context of ageing and chronic disease-related accelerated ageing. Previous research by our group has highlighted the anti-oxidant and anti-inflammatory potential of grape-derived polyphenols in the context of acute inflammation and oxidative stress. The aim here was to add to this by assessing efficacy of the treatment (acutely) to address ageing-associated cumulative pro-oxidant and pro-inflammatory changes in an in vitro model. Blood from young and aged humans was analysed for baseline oxidative stress and inflammatory status. Isolated neutrophils were acutely exposed to the polyphenol treatment in vitro. The chemokinetic capacity of treated and control neutrophils in response to fMLP was subsequently determined in a Dunn chamber, using live cell imaging. Neutrophils were also analysed for the expression of selected molecular markers associated with functional capacity and oxidative stress. Results indicate that the aged population had significantly worse oxidative stress and inflammatory profiles (higher plasma conjugated dienes and MPO) than young controls. Neutrophils isolated from both young and aged individuals had improved chemokinetic accuracy and capacity after in vitro polyphenol treatment. Additionally, increased shedding of CD16 and expression of CD66b suggested sites via which the polyphenol achieved improved neutrophil motility. We conclude that grape seed-derived polyphenols facilitated improved neutrophil functionality by acting on the molecular targets elucidated here.     

基于抗菌药物分级管理的我院抗菌药物医嘱评价

摘 要 目的：明确我院三级抗菌药物（非限制使用级、限制使用级、特殊使用级）不合理使用情况的特点及差异,为临床规范使用抗菌药物提供理论依据。 方法：对我院736份出院病历的1 228条抗菌药物医嘱逐条进行合理性点评,并对三级抗菌药物医嘱不合理使用率、分布特点、类型进行比较。 结果：我院三级抗菌药物不合理使用率差异不全相同,以限制使用级为最高（25.0%）,特殊使用级最低（9.8%）,两者比较差异有统计学意义（P<0.01）。以头孢菌素为主的β内酰胺类抗菌药物的不合理医嘱最多。三级抗菌药物不合理用药类型差异有统计学意义（P<0.05）。“抗菌药物用法用量不适宜”、“围手术期预防性使用抗菌药物不合理”为非限制使用级及限制使用级抗菌药物的主要不合理医嘱构成。而“遴选抗菌药物不适宜”则为特殊使用级抗菌药物的主要不合理医嘱构成。结论：我院抗菌药物不合理使用情况较普遍。医院需针对各级抗菌药物使用中存在的主要问题加强抗菌药物的管理。     

A new strategy for choosing “Q-markers” via network pharmacology,application to the quality control of a Chinese medical preparation

Due to its chemical complexity, proper quality control for a Chinese medical preparation (CMP) has been a great challenge. Choosing the appropriate quality markers (Q-markers) for quality control of CMP is an important work. Best of all, the chosen Q-markers are the main chemical compounds from the herbals as well as the active constituents of this CMP. Only in this way the established quality control system can really achieve the purpose of controlling the quality of CMP and ensuring the safely and effectively use of CMP. To achieve the purpose, network pharmacology combined with the contents of chemical compounds in the CMP has been used in this research. We took an anti-arrhythmic CMP, Shenxian-Shengmai oral liquid (SSOL), as an example. Firstly, UPLC-QTOF-MS/MS method was used to analyze the main components of SSOL. A total of 64 compounds were unambiguously or tentatively identified and 32 of them were further validated by reference compounds. Secondly, the network was constructed based on the identified compounds to predict the effective compounds related to cardiac arrhythmias. Based on the existing database and the operation method of topology, a method of double network analysis (DNAA) was proposed, from which 10 important targets in the pathway of arrhythmia were screened out, and 26 compounds had good antiarrhythmic activity. Based on the prediction results of network pharmacology along with the contents of the compounds in this CMP, ten representative compounds were chosen as the Q-markers for the quality control of SSOL. We find that five of these ten compounds, including danshensu, rosmarinic acid, salvianolic acid A, epimedin A and icariin, have antiarrhythmic activity. Then, the UPLC-DAD method was established as the control method for SSOL.     

Two flavonol glycosides from <Emphasis Type="Italic">Liparis bootanensis</Emphasis>

Two new flavonol glycosides, bootanenside I and II (1 and 2), along with ten known compounds (3–12), were isolated from whole plant of Liparis bootanensis Griff. Their structures were elucidated on the basis of extensive spectroscopic analyses, including high-resolution electrospray-ionization mass spectrometry (HR–ESIMS) and one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR). The cytotoxicity of the compounds was investigated against HCT116 human cancer cell line, revealing that none of them possessed considerable cytotoxic activity. Bioassays of the new metabolites showed that compounds 1 and 2 displayed moderate in vitro antiinflammatory activity by inhibiting expression of inducible nitric oxide synthase (iNOS) protein in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.     

Enzyme linked immunosorbent assay for total potent estrogenic miroestrol and deoxymiroestrol of <Emphasis Type="Italic">Pueraria candollei</Emphasis>, a Thai herb for menopause remedy

Miroestrol and deoxymiroestrol are the most potent phytoestrogens of Pueraria candollei var. mirifica, having been proved as an effective herb for menopausal symptoms in folk medicines and clinical trials. To ensure efficacy and safety of P. candollei var. mirifica involved in nutraceutical products being available worldwide, the content of potent phytoestrogens as active ingredients should be specified. Therefore, in this study, we produced a monoclonal antibody for total analysis of potent estrogenic miroestrol and deoxymiroestrol, for which an analytical method was developed using a procedure for an indirect competitive enzyme-linked immunosorbent assay. The antibody exhibited equal reactivity against miroestrol and deoxymiroestrol. The sensitivity of determination was in the range of 31.3–500 ng/ml with high precision. The analytical parameters, such as accuracy (99.6–106% recovery) and high correlation with a HPLC–UV method, indicated the reliability of analysis. This method is of high performance, and it is cheap to control optimal miroestrol and deoxymiroestrol doses of P. candollei var. mirifica nutraceutical products.     

Total syntheses of schizandriside,saracoside and (±)-isolariciresinol with antioxidant activities

Lignans are widely distributed in plants and exhibit significant pharmacological effects, including anti-tumor and antioxidative activities. Here, we describe the total synthesis of schizandriside (1), a compound we previously isolated from Saraca asoca by monitoring antioxidative activity using the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Starting from a tandem Michael-aldol reaction, the lignan skeleton was synthesized in 6 steps, including a cyclization step. To determine the stereochemistry of 1, we synthesized the natural product (±)-isolariciresinol (18) from alcohol 17. Comparison of the spectral data showed good agreement. Glycosylation was investigated using four different glycosyl donors. Only the Koenigs–Knorr condition using silver trifluoromethanesulfonate with 1,1,3,3-tetramethylurea provided the glycosylated product. Deprotection and purification using reverse-phase high-performance liquid chromatography gave schizandriside (1) and its diastereomer saracoside (2). Synthesized 1, 2 and 18 showed antioxidant activity with IC₅₀?=?34.4, 28.8, 53.0 μM, respectively.