Journal of Pharmaceutical Analysis

Small molecule inhibitors of RORγt for Th17 regulation in inflammatory and autoimmune diseases

Jiuping Zeng, Mingxing Li, Qianyun Zhao, Meijuan Chen, Long Zhao, Shulin Wei, Huan Yang, Yueshui Zhao, Anqi Wang, Jing Shen, Fukuan Du, Yu Chen, Shuai Deng, Fang Wang, Zhuo Zhang, Zhi Li, Tiangang Wang, Shengpeng Wang, Zhangang Xiao, Xu Wu

2023, 13(6): 545-562. doi: 10.1016/j.jpha.2023.05.009

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Abstract:
As a ligand-dependent transcription factor, retinoid-associated orphan receptor γt (RORγt) that controls T helper (Th) 17 cell differentiation and interleukin (IL)-17 expression plays a critical role in the progression of several inflammatory and autoimmune conditions. An emerging novel approach to the therapy of these diseases thus involves controlling the transcriptional capacity of RORγt to decrease Th17 cell development and IL-17 production. Several RORγt inhibitors including both antagonists and inverse agonists have been discovered to regulate the transcriptional activity of RORγt by binding to orthosteric- or allosteric-binding sites in the ligand-binding domain. Some of small-molecule inhibitors have entered clinical evaluations. Therefore, in current review, the role of RORγt in Th17 regulation and Th17-related inflammatory and autoimmune diseases was highlighted. Notably, the recently developed RORγt inhibitors were summarized, with an emphasis on their optimization from lead compounds, efficacy, toxicity, mechanisms of action, and clinical trials. The limitations of current development in this area were also discussed to facilitate future research.

Novel wine in an old bottle: Preventive and therapeutic potentials of andrographolide in atherosclerotic cardiovascular diseases

Tingting Gou, Minghao Hu, Min Xu, Yuchen Chen, Rong Chen, Tao Zhou, Junjing Liu, Li Guo, Hui Ao, Qiang Ye

2023, 13(6): 563-589. doi: 10.1016/j.jpha.2023.05.010

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Abstract:
Atherosclerotic cardiovascular disease (ASCVD) frequently results in sudden death and poses a serious threat to public health worldwide. The drugs approved for the prevention and treatment of ASCVD are usually used in combination but are inefficient owing to their side effects and single therapeutic targets. Therefore, the use of natural products in developing drugs for the prevention and treatment of ASCVD has received great scholarly attention. Andrographolide (AG) is a diterpenoid lactone compound extracted from Andrographis paniculata. In addition to its use in conditions such as sore throat, AG can be used to prevent and treat ASCVD. It is different from drugs that are commonly used in the prevention and treatment of ASCVD and can not only treat obesity, diabetes, hyperlipidaemia and ASCVD but also inhibit the pathological process of atherosclerosis (AS) including lipid accumulation, inflammation, oxidative stress and cellular abnormalities by regulating various targets and pathways. However, the pharmacological mechanisms of AG underlying the prevention and treatment of ASCVD have not been corroborated, which may hinder its clinical development and application. Therefore, this review summarizes the physiological and pathological mechanisms underlying the development of ASCVD and the in vivo and in vitro pharmacological effects of AG on the relative risk factors of AS and ASCVD. The findings support the use of the old pharmacological compound (‘old bottle’) as a novel drug (‘novel wine’) for the prevention and treatment of ASCVD. Additionally, this review summarizes studies on the availability as well as pharmaceutical and pharmacokinetic properties of AG, aiming to provide more information regarding the clinical application and further research and development of AG.

Inhibition of histone methyltransferase PRMT5 attenuates cisplatin-induced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway

Zhiwei Zheng, Benyu Nan, Chang Liu, Dongmei Tang, Wen Li, Liping Zhao, Guohui Nie, Yingzi He

2023, 13(6): 590-602. doi: 10.1016/j.jpha.2023.04.014

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Abstract:
This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5 (PRMT5) in cisplatin-induced hearing loss. The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry, apoptosis assays, and auditory brainstem response. The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction (CUT&Tag-qPCR) analyses in the HEI-OC1 cell line. Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species. CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene, thus activating the expression of Pik3ca. These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatin-induced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

Cocktail hepatocarcinoma therapy by a super-assembled nano-pill targeting XPO1 and ATR synergistically

Liuyun Gong, Yinliang Lu, Jing Wang, Xinyue Li, Jing Zhao, Yuetong Chen, Rongze Ma, Jinlu Ma, Tianya Liu, Suxia Han

2023, 13(6): 603-615. doi: 10.1016/j.jpha.2023.04.017

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Abstract:
Intensive cancer treatment with drug combination is widely exploited in the clinic but suffers from inconsistent pharmacokinetics among different therapeutic agents. To overcome it, the emerging nanomedicine offers an unparalleled opportunity for encapsulating multiple drugs in a nano-carrier. Herein, a two-step super-assembled strategy was performed to unify the pharmacokinetics of a peptide and a small molecular compound. In this proof-of-concept study, the bioinformatics analysis firstly revealed the potential synergies towards hepatoma therapy for the associative inhibition of exportin 1 (XPO1) and ataxia telangiectasia mutated-Rad3-related (ATR), and then a super-assembled nano-pill (gold nano drug carrier loaded AZD6738 and 97−110 amino acids of apoptin (AP) (AA@G)) was constructed through camouflaging AZD6738 (ATR small-molecule inhibitor)-binding human serum albumin onto the AP-Au supramolecular nanoparticle. As expected, both in vitro and in vivo experiment results verified that the AA@G possessed extraordinary biocompatibility and enhanced therapeutic effect through inducing cell cycle arrest, promoting DNA damage and inhibiting DNA repair of hepatoma cell. This work not only provides a co-delivery strategy for intensive liver cancer treatment with the clinical translational potential, but develops a common approach to unify the pharmacokinetics of peptide and small-molecular compounds, thereby extending the scope of drugs for developing the advanced combination therapy.

Combination immunotherapy of glioblastoma with dendritic cell cancer vaccines, anti-PD-1 and poly I:C

Ping Zhu, Shi-You Li, Jin Ding, Zhou Fei, Sheng-Nan Sun, Zhao-Hui Zheng, Ding Wei, Jun Jiang, Jin-Lin Miao, San-Zhong Li, Xing Luo, Kui Zhang, Bin Wang, Kun Zhang, Su Pu, Qian-Ting Wang, Xin-Yue Zhang, Gao-Liu Wen, Jun O. Liu, John Thomas August, Huijie Bian, Zhi-Nan Chen, You-Wen He

2023, 13(6): 616-624. doi: 10.1016/j.jpha.2023.04.012

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Abstract:
Glioblastoma (GBM) is a lethal cancer with limited therapeutic options. Dendritic cell (DC)-based cancer vaccines provide a promising approach for GBM treatment. Clinical studies suggest that other immunotherapeutic agents may be combined with DC vaccines to further enhance antitumor activity. Here, we report a GBM case with combination immunotherapy consisting of DC vaccines, anti-programmed death-1 (anti-PD-1) and poly I:C as well as the chemotherapeutic agent cyclophosphamide that was integrated with standard chemoradiation therapy, and the patient remained disease-free for 69 months. The patient received DC vaccines loaded with multiple forms of tumor antigens, including mRNA-tumor associated antigens (TAA), mRNA-neoantigens, and hypochlorous acid (HOCl)-oxidized tumor lysates. Furthermore, mRNA-TAAs were modified with a novel TriVac technology that fuses TAAs with a destabilization domain and inserts TAAs into full-length lysosomal associated membrane protein-1 to enhance major histocompatibility complex (MHC) class I and II antigen presentation. The treatment consisted of 42 DC cancer vaccine infusions, 26 anti-PD-1 antibody nivolumab administrations and 126 poly I:C injections for DC infusions. The patient also received 28 doses of cyclophosphamide for depletion of regulatory T cells. No immunotherapy-related adverse events were observed during the treatment. Robust antitumor CD4⁺ and CD8⁺ T-cell responses were detected. The patient remains free of disease progression. This is the first case report on the combination of the above three agents to treat glioblastoma patients. Our results suggest that integrated combination immunotherapy is safe and feasible for long-term treatment in this patient. A large-scale trial to validate these findings is warranted.

Effects and translatomics characteristics of a small-molecule inhibitor of METTL3 against non-small cell lung cancer

Han Xiao, Rong Zhao, Wangyang Meng, Yongde Liao

2023, 13(6): 625-639. doi: 10.1016/j.jpha.2023.04.009

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Abstract:
In non-small cell lung cancer (NSCLC), the heterogeneity promotes drug resistance, and the restricted expression of programmed death-ligand 1 (PD-L1) limits the immunotherapy benefits. Based on the mechanisms related to translation regulation and the association with PD-L1 of methyltransferase-like 3 (METTL3), the novel small-molecule inhibitor STM2457 is assumed to be useful for the treatment of NSCLC. We evaluated the efficacy of STM2457 in vivo and in vitro and confirmed the effects of its inhibition on disease progression. Next, we explored the effect of STM2457 on METTL3 and revealed its effects on the inhibition of catalytic activity and upregulation of METTL3 protein expression. Importantly, we described the genome-wide characteristics of multiple omics data acquired from RNA sequencing, ribosome profiling, and methylated RNA immunoprecipitation sequencing data under STM2457 treatment or METTL3 knockout. We also constructed a model for the regulation of the translation of METTL3 and PD-L1. Finally, we found PD-L1 upregulation by STM2457 in vivo and in vitro. In conclusion, STM2457 is a potential novel suppressor based on its inhibitory effect on tumor progression and may be able to overcome the heterogeneity based on its impact on the translatome. Furthermore, it can improve the immunotherapy outcomes based on PD-L1 upregulation in NSCLC.

Radix Paeoniae Alba attenuates Radix Bupleuri-induced hepatotoxicity by modulating gut microbiota to alleviate the inhibition of saikosaponins on glutathione synthetase

Congcong Chen, Wenxia Gong, Junshen Tian, Xiaoxia Gao, Xuemei Qin, Guanhua Du, Yuzhi Zhou

2023, 13(6): 640-659. doi: 10.1016/j.jpha.2023.04.016

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Abstract:
Radix Bupleuri (RB) is commonly used to treat depression, but it can also lead to hepatotoxicity after long-term use. In many anti-depression prescriptions, RB is often used in combination with Radix Paeoniae Alba (RPA) as an herb pair. However, whether RPA can alleviate RB-induced hepatotoxicity remain unclear. In this work, the results confirmed that RB had a dose-dependent antidepressant effect, but the optimal antidepressant dose caused hepatotoxicity. Notably, RPA effectively reversed RB-induced hepatotoxicity. Afterward, the mechanism of RB-induced hepatotoxicity was confirmed. The results showed that saikosaponin A and saikosaponin D could inhibit GSH synthase (GSS) activity in the liver, and further cause liver injury through oxidative stress and nuclear factor kappa B (NF-κB)/NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathway. Furthermore, the mechanisms by which RPA attenuates RB-induced hepatotoxicity were investigated. The results demonstrated that RPA increased the abundance of intestinal bacteria with glycosidase activity, thereby promoting the conversion of saikosaponins to saikogenins in vivo. Different from saikosaponin A and saikosaponin D, which are directly combined with GSS as an inhibitor, their deglycosylation conversion products saikogenin F and saikogenin G exhibited no GSS binding activity. Based on this, RPA can alleviate the inhibitory effect of saikosaponins on GSS activity to reshape the liver redox balance and further reverse the RB-induced liver inflammatory response by the NF-κB/NLRP3 pathway. In conclusion, the present study suggests that promoting the conversion of saikosaponins by modulating gut microbiota to attenuate the inhibition of GSS is the potential mechanism by which RPA prevents RB-induced hepatotoxicity.

Quantitative assessment of the relevance of organic-anion-transporting-polypeptide 1B1 and 2B1 polymorphisms in fexofenadine pharmacokinetic variants via pharmacometrics

Ji-Hun Jang, Seung-Hyun Jeong, Yong-Bok Lee

2023, 13(6): 660-672. doi: 10.1016/j.jpha.2023.04.001

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Abstract:
Fexofenadine is useful in various allergic disease treatment. However, the pharmacokinetic variability information and quantitative factor identification of fexofenadine are very lacking. This study aimed to verify the validity of previously proposed genetic factors through fexofenadine population pharmacokinetic modeling and to explore the quantitative correlations affecting the pharmacokinetic variability. Polymorphisms of the organic-anion-transporting-polypeptide (OATP) 1B1 and 2B1 have been proposed to be closely related to fexofenadine pharmacokinetic diversity. Therefore, modeling was performed using fexofenadine oral exposure data according to the OATP1B1- and 2B1-polymorphisms. OATP1B1 and 2B1 were identified as effective covariates of clearance (CL/F) and distribution volume (V/F)-CL/F, respectively, in fexofenadine pharmacokinetic variability. CL/F and average steady-state plasma concentration of fexofenadine differed by up to 2.17- and 2.20-folds, respectively, depending on the OATP1B1 polymorphism. Among the individuals with different OATP2B1 polymorphisms, the CL/F and V/F differed by up to 1.73- and 2.00-folds, respectively. Ratio of the areas under the curves following single- and multiple-administrations, and the cumulative ratio were significantly different between OATP1B1- and 2B1-polymorphism groups. Based on quantitative prediction comparison through a model-based approach, OATP1B1 was confirmed to be relatively more important than 2B1 regarding the degree of effect on fexofenadine pharmacokinetic variability. Based on the established pharmacokinetic-pharmacodynamic relationship, the difference in fexofenadine efficacy according to genetic polymorphisms of OATP1B1 and 2B1 was 1.25- and 0.87-times, respectively, and genetic consideration of OATP1B1 was expected to be important in the pharmacodynamics area as well. This population pharmacometrics study will be a very useful starting point for fexofenadine precision medicine.

Tumor cell membrane-coated continuous electrochemical sensor for GLUT1 inhibitor screening

Jiaqian Zhao, Yuqiao Liu, Ling Zhu, Junmin Li, Yanhui Liu, Jiarui Luo, Tian Xie, Dajing Chen

2023, 13(6): 673-682. doi: 10.1016/j.jpha.2023.04.015

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Abstract:
Glucose transporter 1 (GLUT1) overexpression in tumor cells is a potential target for drug therapy, but few studies have reported screening GLUT1 inhibitors from natural or synthetic compounds. With current analysis techniques, it is difficult to accurately monitor the GLUT1 inhibitory effect of drug molecules in real-time. We developed a cell membrane-based glucose sensor (CMGS) that integrated a hydrogel electrode with tumor cell membranes to monitor GLUT1 transmembrane transport and screen for GLUT1 inhibitors in traditional Chinese medicines (TCMs). CMGS is compatible with cell membranes of various origins, including different types of tumors and cell lines with GLUT1 expression knocked down by small interfering RNA or small molecules. Based on CMGS continuous monitoring technique, we investigated the glucose transport kinetics of cell membranes with varying levels of GLUT1 expression. We used CMGS to determine the GLUT1-inhibitory effects of drug monomers with similar structures from Scutellaria baicalensis and catechins families. Results were consistent with those of the cellular glucose uptake test and molecular-docking simulation. CMGS could accurately screen drug molecules in TCMs that inhibit GLUT1, providing a new strategy for studying transmembrane protein-receptor interactions.

Identification, structure elucidation and origin of a common pyridinium-thiocyanate intermediate in electrospray mass spectrometry among the benziamidazole-class proton pump inhibitors

Dong Sun, Chunyu Wang, Yanxia Fan, Jingkai Gu

2023, 13(6): 683-688. doi: 10.1016/j.jpha.2023.04.011

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Abstract:
During the analysis of benziamidazole-class irreversible proton pump inhibitors, an unusual mass spectral response with the mass-to-charge ratio at [M+10]⁺ intrigued us, as it couldn't be assigned to any literature known relevant structure, intermediate or adduct ion. Moreover, this mysterious mass pattern of [M+10]⁺ has been gradually observed by series of marketed proton pump inhibitors, viz. omeprazole, pantoprazole, lansoprazole and rabeprazole. All the previous attempts to isolate the corresponding component were unsuccessful. The investigation of present work addresses this kind of signal to a pyridinium thiocyanate mass spectral intermediate (10) , which is the common fragment ion of series of labile aggregates. The origin of such aggregates can be traced to the reactive intermediates formed by acid-promoted degradation. These reactive intermediates tend to react with each other and give raise series of complicated aggregates systematically in a water/acetonitrile solution by electrospray ionization. The structure of the corresponding pyridinium thiocyanate species of omeprazole ( 10a ) has been eventually characterized with the help of synthetic specimen ( 10a′ ). Our structural proposal as well as its origin was supported by in situ nuclear magnetic resonance, chemical derivatization and colorimetric experiments.

2023 Vol. 13, No. 6