Evaluating the viral clearance ability of continuous monoclonal antibody purification steps, in order to inactivate and/or remove four model viruses.

Background and Objectives: Viral clearance studies are an essential part of a manufacturer's plan to ensure the safety of an injectable biologic product. In this way, viral safety is a critical quality attribute for biologics such as monoclonal antibodies (Mabs). Evaluation of virus purification by downstream processes is a key component of risk mitigation. In this study, the capability of continuous monoclonal antibody purification steps was evaluated in the process of instant monoclonal antibody purification in different stages of purification, and the amount of reduction or inactivation of each step was determined. Materials and Methods: Four enveloped and non-enveloped viral models VSV, Reovirus, EMCV, and HSV1 were used for spiking in selected samples in the designated tests, to have a comprehensive examination of the ability to clear the virus such as the type of genetic material, chemical resistance, and particle size. A TCID50 and qPCR methods were used to measure viral reduction. Two cell lines, Vero (African green monkey kidney) and L929 (Mouse fibroblast) were used for 4 model viruses propagation. The steps that were evaluated included 4 steps monoclonal antibody purification; cation exchange chromatography, acidic pH treatment, affinity chromatography, and nanofiltration. Results: The nano-filter stage showed the highest viral reduction and cation exchange chromatography showed the lowest reduction. The cumulative decrease using TCID50 is equal to 19.27 [log10] for all steps and for the qPCR method is equal to 12.47 [log10] in three steps of nano-filter, affinity chromatography, and ion exchange chromatography. Conclusion: The overall average reduction coefficient for all four model viruses is significantly high, which indicates the high capacity of the monoclonal antibody production process in inactivating and removing viruses leads to reducing the load of all four model viruses.

Screening of Cyclodextrins in the Processing of Buserelin Dry Powders for Inhalation Prepared by Spray Freeze-Drying.

Purpose: In this study, we prepared inhalable buserelin microparticles using the spray freeze-drying (SFD) method for pulmonary drug delivery. Raffinose as a cryoprotectant carrier was combined with two levels of five different cyclodextrins (CDs) and then processed by SFD. Methods: Dry powder diameters were evaluated by laser light scattering and morphology was determined by scanning electron microscopy (SEM). Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis were utilized for the determination of crystalline structures. The aerodynamic properties of the spray freeze-dried powders were evaluated by twin stage impinger (TSI) and the stability of prepared samples was assessed under normal and accelerated conditions. Results: The prepared powders were mostly porous spheres and the size of microparticles ranged from 9.08 to 13.53 µm, which are suitable as spray-freeze dried particles. All formulations showed amorphous structure confirmed by DSC and XRD. The aerosolization performance of the formulation containing buserelin, raffinose and 5% beta-cyclodextrin (ß-CD), was the highest and its fine particle fraction (FPF) was 69.38%. The more circular and separated structures were observed in higher concentrations of CDs, which were compatible with FPFs. The highest stability was obtained in the formulation containing hydroxypropyl beta-cyclodextrin (HP-ß-16. CD) 5%. On the contrary, sulfobutylether beta-cyclodextrin (SBE-ß-CD) 5% bearing particles showed the least stability. Conclusion: By adjusting the type and ratio of CDs in the presence of raffinose, the prepared formulations could effectively enhance the aerosolization and stability of buserelin. Therefore, they can be proposed as a suitable career for lung drug delivery.

Design, synthesis, and cytotoxic evaluation of quinazoline derivatives bearing triazole-acetamides.

A novel series of quinazoline-based agents bearing triazole-acetamides 8a-l were designed and synthesized. All the obtained compounds were tested for in vitro cytotoxic activities against three human cancer cell lines named HCT-116, MCF-7, and HepG2, as well as a normal cell line WRL-68 after 48 and 72 h. The results implied that quinazoline-oxymethyltriazole compounds exhibited moderate to good anticancer potential. The most potent derivative against HCT-116 was 8a (X = 4-OCH3 and R = H) with IC50 values of 10.72 and 5.33 µM after 48 and 72 h compared with doxorubicin with IC50 values of 1.66 and 1.21 µM, respectively. The same trend was seen in the HepG2 cancerous cell line in which 8a recorded the best results with IC50 values of 17.48 and 7.94 after 48 and 72 h, respectively. The cytotoxic analysis against MCF-7 showed that 8f with IC50 = 21.29 µM (48 h) exhibited the best activity, while compounds 8k (IC50 = 11.32 µM) and 8a (IC50 = 12.96 µM), known as the most effective cytotoxic agents after 72 h. Doxorubicin as positive control exhibited IC50 values of 1.15 and 0.82 µM after 48 and 72 h, respectively. Noteworthy, all derivatives showed limited toxicity against the normal cell line. Moreover, docking studies were also presented to understand the interactions between these novel derivatives and possible targets.

Assessing the ergonomic status of commonly used chairs in Iran's universities based on combinational equations and anthropometric indices.

BACKGROUND: Inappropriate design of chairs and disproportion with students' body dimensions can influence their physical and mental health as well as their educational efficiency and concentration. OBJECTIVE: The aim of this study was to examine the ergonomic status of commonly used chairs in Iran's universities using combinational equations and anthropometric indices. METHODS: Participants in this cross-sectional study were 166 students in Iran. Eleven anthropometric parameters of the students and nine dimensions in seven types of commonly used chairs in Iran's universities were measured. Combinational equations were used to determine the fitness of the chairs. RESULTS: There was a major mismatch between most students' anthropometric measurements and the dimensions of the chairs. Backrest height and seat height were the best and the worst features, respectively, according to ergonomic recommendations for chairs. The recommended measurements of seat height, seat depth, seat width, desk height, desk width, desk length, backrest width, backrest height, and desk distance were 332, 420, 436, 245, 95, 511, 426, 550, and 281 mm, respectively. CONCLUSION: None of the commonly used chairs in Iran's universities were found to be a good fit to the students' anthropometric dimensions. Therefore, in order to prevent inappropriate body postures, chair designs have to reviewed, and made to fit with Iranian students' anthropometric data. The recommended measurements obtained in this study can be used to design a suitable ergonomic chair to match with a high percentage of Iranian students.

Differentiation of multipotent stem cells to insulin-producing cells for treatment of diabetes mellitus: bone marrow- and adipose tissue-derived cells comparison.

BACKGROUND: Mesenchymal stem cells (MSCs) from human adipose tissue and bone marrow have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose and bone marrow tissue into cells with a pancreatic endocrine phenotype and to compare the potency of these cells together. METHODS AND RESULTS: MSCs were pre-induced with nicotinamide, mercaptoethanol, B-27 and b-FGF in L-DMEM for 2 days and re-induced again in supplemented H-DMEM for another 3 days. Expression of five genes in differentiated beta cells was evaluated by Real-time PCR and western blotting and the potency of insulin release in response to glucose stimulation was evaluated by insulin and C-peptide ELISA kit. The differentiated cells were evaluated by immunocytochemistry staining for Insulin and PDX-1. Quantitative RT-PCR results showed up-regulation of four genes in differentiated beta-islet cells (Insulin, Ngn-3, Pax-4 and Pdx-1) compared with the control. Western blot analysis showed that MSCs cells mainly produced proinsulin and insulin after differentiation but nestin was more expressed in pre-differentiated stem cells. Glucose and insulin secretion assay showed that insulin levels and C-peptide secretion were significantly increased in response to 10 mM glucose. CONCLUSIONS: Our study showed that both adipose and bone marrow stem cells could differentiate into functional beta-islet cells but it seems that adipose stem cells could be a better choice for treatment of diabetes mellitus according to their higher potency.

Design, Synthesis, in Vitro, and in Silico Evaluation of N-Phenylacetamide-Oxindole-Thiosemicarbazide Hybrids as New Potential Tyrosinase Inhibitors.

A novel series of N-phenylacetamide-oxindole-thiosemicarbazide hybrids were synthesized and evaluated for their tyrosinase inhibitory activity. According to tyrosinase inhibition results, all the synthesized compounds showed high tyrosinase inhibitory activity with IC50 values ranging from 0.8 to 3.88 µM in comparison to positive control kojic acid with IC50 value of 36.32 µM. Among tested compounds, analog 7o, containing the 2-methyl-4-nitrophenyl on N-phenylacetamide moiety displayed superior tyrosinase inhibition. This compound was around 45-fold more potent than kojic acid. The kinetic analysis of compound 7o demonstrated that this compound is a competitive inhibitor against tyrosinase. Docking study of this compound demonstrated that compound 7o interacted with critical histidine residues within tyrosinase active site.

Design, synthesis, and α-glucosidase-inhibitory activity of phenoxy-biscoumarin-N-phenylacetamide hybrids.

Thirteen new phenoxy-biscoumarin-N-phenylacetamide derivatives (7a-m) were designed based on a molecular hybridization approach as new α-glucosidase inhibitors. These compounds were synthesized with high yields and evaluated in vitro for their inhibitory activity against yeast α-glucosidase. The obtained results revealed that a significant proportion of the synthesized compounds showed considerable α-glucosidase-inhibitory activity in comparison to acarbose as a positive control. Representatively, 2-(4-(bis(4-hydroxy-2-oxo-2H-chromen-3-yl)methyl)phenoxy)-N-(4-bromophenyl)acetamide (7f), with IC50 = 41.73 ± 0.38 µM against α-glucosidase, was around 18 times more potent than acarbose (IC50 = 750.0 ± 10.0 µM). This compound was a competitive α-glucosidase inhibitor. Molecular modeling and dynamic simulation of these compounds confirmed the obtained results through in vitro experiments. Prediction of the druglikeness/ADME/toxicity of the compound 7f and comparison with the standard drug acarbose showed that the new compound 7f was probably better than the standard drug in terms of toxicity.

An Integrated Strategy Reveals Complex Glycosylation of Erythropoietin Using Mass Spectrometry.

The characterization of therapeutic glycoproteins is challenging due to the structural heterogeneity of the therapeutic protein glycosylation. This study presents an in-depth analytical strategy for glycosylation of first-generation erythropoietin (epoetin beta), including a developed mass spectrometric workflow for N-glycan analysis, bottom-up mass spectrometric methods for site-specific N-glycosylation, and a LC-MS approach for O-glycan identification. Permethylated N-glycans, peptides, and enriched glycopeptides of erythropoietin were analyzed by nanoLC-MS/MS, and de-N-glycosylated erythropoietin was measured by LC-MS, enabling the qualitative and quantitative analysis of glycosylation and different glycan modifications (e.g., phosphorylation and O-acetylation). The newly developed Python scripts enabled the identification of 140 N-glycan compositions (237 N-glycan structures) from erythropoietin, especially including 8 phosphorylated N-glycan species. The site-specificity of N-glycans was revealed at the glycopeptide level by pGlyco software using different proteases. In total, 114 N-glycan compositions were identified from glycopeptide analysis. Moreover, LC-MS analysis of de-N-glycosylated erythropoietin species identified two O-glycan compositions based on the mass shifts between non-O-glycosylated and O-glycosylated species. Finally, this integrated strategy was proved to realize the in-depth glycosylation analysis of a therapeutic glycoprotein to understand its pharmacological properties and improving the manufacturing processes.

Recent Opportunities and Challenges in Selective C-H Functionalization of Methyl Azaarenes: A Highlight from 2010 to 2020 Studies.

Azaarenes are unique scaffolds that are frequently found in pharmaceuticals. Herein we have summarized the recent synthetic available methods in C-H functionalization of methylazaarenes from 2010 to 2020. Multiple approaches involving halogenation, alkylation via different methods, alkenylation, oxidative functionalization, and cyclization of the methylazaarenes will be discussed in this review.

Vinylazides: versatile synthons and magical precursors for the construction of N-heterocycles.

Nowadays, application of vinylazides as precursors is a key method for the construction of N-heterocycles in organic synthesis. These versatile three-atom synthons can be converted into intermediates such as 2H-azirines, iminyl radicals, iminyl metal complexes, iminyl inions and nitrilium ions that subsequently afford a wide range of polyfunctional cyclic nitrogen-containing compounds. In this review, the reactions of vinylazides leading to these products (in the last decade) are categorized based on the types of the resulting N-heterocyclic rings and a brief and concise description of the reaction mechanisms is presented.

Recent strategies in the synthesis of thiophene derivatives: highlights from the 2012-2020 literature.

Thiophene-based analogs have been fascinated by a growing number of scientists as a potential class of biologically active compounds. Furthermore, they play a vital role for medicinal chemists to improve advanced compounds with a variety of biological effects. The current review envisioned to highlight some recent and particularly remarkable examples of the synthesis of thiophene derivatives by heterocyclization of various substrates from 2012 on.

Synthesis and pharmacological properties of polysubstituted 2-amino-4H-pyran-3-carbonitrile derivatives.

2-Amino-3-cyano-4H-chromenes are structural core motifs that received increasing attention in the last years due to their interesting potential pharmacological properties. In this review, the synthetic methods for these compounds are classified based on the type of catalyst in the pertinent reactions. In addition, the wide range of pharmacological properties of these compounds is covered in a separate section.

The use of magnetic starch as a support for an ionic liquid-ß-cyclodextrin based catalyst for the synthesis of imidazothiadiazolamine derivatives.

In this paper, a novel catalyst is introduced based on the introduction of an ionic liquid onto the ß-cyclodextrin. The ionic liquid-ß-cyclodextrin was anchored to magnetic starch (denoted ßCD-IL@M-Starch) and fully characterized by several methods including TEM, TGA, VSM and FT-IR. The catalyst was used for the synthesis of imidazo[2,1-b][1,3,4]thiadiazol-5-amine and imidazo[1,2-a]pyridin-3-amine derivatives. ßCD-IL@M-Starch catalyst showed very good activity in the synthesis of diphenylimidazo[2,1-b][1,3,4]thiadiazol-5-amine derivatives from the corresponding benzaldehyde, semicarbazide, benzaldehydes and isocyanides. The products were obtained in a mild reaction conditions in good isolated yields in the presence of ßCD-IL@M-Starch as catalyst. The catalyst showed to be magnetically reusable, and gave very good results in 10 sequential reactions.