Identification of sorbitol esterification of glutamic acid by LC-MS/MS in a monoclonal antibody stability assessment.

The stability of monoclonal antibodies (mAbs) is vital for their therapeutic success. Sorbitol, a common mAb stabilizer used to prevent aggregation, was evaluated for any potential adverse effects on the chemical stability of mAb X. An LC-MS/MS based analysis focusing on the post-translational modifications (PTMs) of mAb X was conducted on samples that had undergone accelerated aging at 40°C. Along with PTMs that are known to affect mAbs' structure function and stability (such as deamidation and oxidation), a novel mAb PTM was discovered, the esterification of glutamic acid by sorbitol. Incubation of mAb X with a 1:1 ratio of unlabeled sorbitol and isotopically labeled sorbitol (13C6) further corroborated that the modification was the consequence of the esterification of glutamic acid by sorbitol. Levels of esterification varied across glutamic acid residues and correlated with incubation time and sorbitol concentration. After 4 weeks of accelerated stability with isotopically labeled sorbitol, it was found that 16% of the total mAb possesses an esterified glutamic acid. No esterification was observed at aspartic acid sites despite the free carboxylic acid side chain. This study unveils a unique modification of mAbs, emphasizing its potential significance for formulation and drug development.

[Cannabinoid receptor 1 agonist arachidonyl-2'-chloroethylamide (ACEA) improves sepsis-associated encephalopathy by inhibiting inflammatory factors].

Objective To investigate the impact of the cannabinoid receptor agonist arachidonyl-2'-chloroethylamide (ACEA) on cognitive function in mice with sepsis-associated encephalopathy (SAE). Methods C57BL/6 mice were randomly divided into artificial cerebrospinal fluid (ACSF) and lipopolysaccharide (LPS) groups. The SAE model was established by intraventricular injection of LPS. The severity of sepsis in mice was assessed by sepsis severity score (MSS) and body mass changes. Behavioral paradigms were used to evaluate motor ability (open field test) and cognitive function (contextual fear conditioning test, Y-maze test). To evaluate the effects of ACEA intervention on SAE, mice were randomly assigned to ACSF group, ACEA intervention combined with ACSF group, LPS group, and ACEA intervention combined with LPS group. The dosage of ACEA intervention was 1.5 mg/kg. Real-time quantitative PCR was used to measure the mRNA expression levels of interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) in mouse hippocampal tissues. Western blot analysis was used to assess the protein levels of IL-6 and TNF-α in the hippocampus. Nissl staining was performed to examine neuronal damage in the CA1 region of the mouse hippocampus. Behavioral paradigms were again employed to evaluate motor ability and cognitive function. Results Three days after intraventricular LPS injection, mice exhibited significant cognitive dysfunction, confirming SAE modeling. Compared to the control group, the LPS group showed significant increases in mRNA of inflammatory factors such as IL-6, TNF-α, and IL-1ß, together with significant increases in IL-6 and TNF-α protein levels in the hippocampus, a decrease in Nissl bodies in the CA1 region, and significant cognitive dysfunction. Compared to the LPS group, the ACEA intervention group showed a significant decrease in the mRNA of IL-6, TNF-α, and IL-1ß, a significant reduction in IL-6 and TNF-α protein levels, an increase in Nissl bodies, and improved cognitive function. Conclusion ACEA improves cognitive function in SAE mice by inhibiting the expression levels of inflammatory factors IL-6 and TNF-α.

The determination of Sulfobutylether ß-Cyclodextrin Sodium (SBECD) by LC-MS/MS and its application in remdesivir pharmacokinetics study for pediatric patients.

SBECD (Captisol®) with an average degree of substitution of 6.5 sulfobutylether functional groups (SBE = 6.5), is a solubility enhancer for remdesivir (RDV) and a major component in Veklury, which was approved by FDA for the treatment of patients with COVID-19 over 12 years old and weighing over 40 kg who require hospitalization. SBECD is cleared mainly by renal filtration, thus, potential accumulation of SBECD in the human body is a concern for patients dosed with Veklury with compromised renal function. An LC-MS/MS method was developed and validated for specific, accurate, and precise determination of SBECD concentrations in human plasma. In this method, the hexa-substituted species, SBE6, was selected for SBECD quantification, and the mass transition from its dicharged molecular ion [(M-2H)/2]2-, Molecular (parent) Ion (Q1)/Molecular (parent) Ion (Q3) of m/z 974.7/974.7, was selected for quantitative analysis of SBECD. Captisol-G (SBE-γ-CD, SBE = 3) was chosen as the internal standard. With 25 µL of formic-acid-treated sample and with a calibration range of 10.0-1000 µg/mL, the method was validated with respect to pre-established criteria based on regulatory guidelines and was applied to determine SBECD levels in plasma samples collected from pediatric patients during RDV clinical studies.

Effect of ginsenoside-Rg1 on experimental Parkinson's disease: A systematic review and meta-analysis of animal studies.

Previous studies have reported that ginsenoside-Rg1 (G-Rg1) was able to mitigate the loss of dopaminergic neurons in animal models of Parkinson's disease (PD). The present study provided a systematic review and meta-analysis of preclinical studies to pool current evidence on the effect of G-Rg1 on neurogenesis in the treatment of PD. Eligible studies were identified through a search from six databases: PubMed, EMBASE, Web of Science, VIP, Chinese National Knowledge Infrastructure and the Wanfang database. Primary outcomes were tyrosine hydroxylase (TH)-positive cells in the nigra, Nissl staining-positive cells in the nigra, pole test time and dopamine (DA) levels in the striatum. A total of 18 eligible studies were identified, involving 343 animals. Of these, 13 reported a significant relationship between G-Rg1 and improved TH-positive cells in the nigra compared with the control group (P<0.00001). Furthermore, 3 studies reported a significant relationship between G-Rg1 and improved Nissl-positive cells in the nigra compared with the control group (P<0.00001). In addition, 4 studies reported a significant effect of G-Rg1 to reduce the total pole test time compared with that in the control group (P=0.001). A total of 3 studies indicated a significant association between G-Rg1 and improved DA levels in the striatum compared with the control group (P<0.00001). These results suggested that G-Rg1 has positive effects in attenuating damage in models of PD, and thus, it is a potential candidate neuroprotective drug for human PD.

Biomimetic Nanosilica-Collagen Scaffolds for In Situ Bone Regeneration: Toward a Cell-Free, One-Step Surgery.

Current approaches to fabrication of nSC composites for bone tissue engineering (BTE) have limited capacity to achieve uniform surface functionalization while replicating the complex architecture and bioactivity of native bone, compromising application of these nanocomposites for in situ bone regeneration. A robust biosilicification strategy is reported to impart a uniform and stable osteoinductive surface to porous collagen scaffolds. The resultant nSC composites possess a native-bone-like porous structure and a nanosilica coating. The osteoinductivity of the nSC scaffolds is strongly dependent on the surface roughness and silicon content in the silica coating. Notably, without the use of exogenous cells and growth factors (GFs), the nSC scaffolds induce successful repair of a critical-sized calvarium defect in a rabbit model. It is revealed that topographic and chemical cues presented by nSC scaffolds could synergistically activate multiple signaling pathways related to mesenchymal stem cell recruitment and bone regeneration. Thus, this facile surface biosilicification approach could be valuable by enabling production of BTE scaffolds with large sizes, complex porous structures, and varied osteoinductivity. The nanosilica-functionalized scaffolds can be implanted via a cell/GF-free, one-step surgery for in situ bone regeneration, thus demonstrating high potential for clinical translation in treatment of massive bone defects.

[ROLE OF TIBIAL TUBERCLE INTERNAL ROTATION TO TREAT RECURRENT PATELLAR DISLOCATION ASSOCIATED WITH TROCHLEAR DYSPLASIA].

OBJECTIVE: To introduce and analyze the role of tibial tubercle internal rotation to treat recurrent patellar dislocation associated with trochlear dysplasia. METHODS: Between February 2007 and April 2011, 28 patients with recurrent patellar dislocation underwent tibial tubercle translocation through medial transfer, rotation and elevation of the tibial tuberosity and the medial patellofemoral ligament (MPFL) reconstruction. There were 4 males and 24 females with an average age of 21.8 years (range, 17-28 years). The disease duration ranged from 6 months to 8 years (mean, 4 years). The patients suffered from 3-10 times patellar dislocation. The result of apprehension test was positive; Lysholm score was (51.64 ± 3.79); Kujala score was (56.89 ± 3.79). According to Dejour classification, 11 cases were rated as type B, 14 cases as type C, and 3 cases as type D; the tibial tuberosity-trocholear distance (TT-TG) was (20.53 ± 2.58) mm; and the patellar tilt angle (PTA) was (29.34 ± 2.54)°. RESULTS: Primary healing of incision was obtained in the others except 1 case of mild infection. Twenty-seven patients were followed up 41.8 months on average (range, 27-74 months). No recurrent dislocation was found, and the result of apprehension test was negative. The knee range of motion restored to normal totally. The postoperative Kujala score and Lysholm score were significantly improved to 88.97 ± 3.06 and 88.95 ± 2.98 (t = -42.005, P = 0.000; t = -43.122, P = 0.000) respectively. TT-TG and PTA restored to normal [(11.77 ± 2.24) mm and (7.99 ± 2.57)°], showing significant differences when compared with preoperative ones(t=13.032, P=0.000; t=29.533, P=0.000). CONCLUSION: The technique of tibial tubercle translocation and MPFL reconstruction is an effective surgical procedure for the treatment of recurrent patellar dislocation associated with trochlear dysplasia. Especially, tibial tubercle internal rotation can improve the patella stability and knee function.

Salt-inducible kinase 1 is involved in high glucose-induced mesangial cell proliferation mediated by the ALK5 signaling pathway.

High glucose levels can induce mesangial cell proliferation and extracellular matrix (ECM) accumulation through the type I activin receptor-like kinase 5 (ALK5) signaling pathway. Salt-inducible kinase 1 (SIK1) prevents fibrosis by downregulating ALK5, while the expression level of the SIK1 protein itself is downregulated by glucose in neuronal cells following ischemia. In this study, we investigated the correlation between SIK1 and the ALK5 signaling pathway in a rat glomerular mesangial cell line (HBZY-1 cells). We found that high glucose levels downregulated the expression level of SIK1 and suppressed the phosphorylation of SIK1 at Thr-182. The downregulation of SIK1 by high glucose was accompanied by the activation of the ALK5 signaling pathway, while the overexpression of SIK1 in the HBZY-1 cells resulted in a decrease in the ALK5 protein level, as well in the levels of its downstream targets, including fibronectin and plasminogen activator inhibitor type I. In conclusion, high glucose may activate the ALK5 signaling pathway by downregulating SIK1, and SIK1 may be a protective factor against cellular proliferation and ECM accumulation in glomerular mesangial cells under high glucose conditions.