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This paper summered the clinical application of Guizhi decoction combined with Guizhigancaolonggumuli decoction in treating one case of insomnia. Based on the Traditional Chinese Medicine syndrome differentiation, Guizhi decoction combined with Guizhigancaolonggumuli decoction was administrated orally, and comprehensive nursing interventions on environment, dietary, exercise, mental health of the patient were carried out to improve the sleep quality of the patient. (本文总结1例采用桂枝汤合桂枝甘草龙骨牡蛎汤加减治疗失眠患者的临床经验。失眠患者多为阴阳不调, 久则易伤阴阳, 故而引起“不寐”。基于中医辨证分型, 桂枝汤合桂枝甘草龙骨牡蛎汤加减, 温补心阳, 调理营卫, 使阴阳调和, 并配合环境、饮食、运动、心理等护理干预, 有效改善了患者睡眠质量。)

Enabled by a plethora of new technologies, research in membrane transporters has exploded in the past decade. The goal of this state-of-the-art article is to describe recent advances in research on membrane transporters that are particularly relevant to drug discovery and development. This review covers advances in basic, translational, and clinical research that has led to an increased understanding of membrane transporters at all levels. At the basic level, we describe the available crystal structures of membrane transporters in both the solute carrier (SLC) and ATP binding cassette superfamilies, which has been enabled by the development of cryogenic electron microscopy methods. Next, we describe new research on lysosomal and mitochondrial transporters as well as recently deorphaned transporters in the SLC superfamily. The translational section includes a summary of proteomic research, which has led to a quantitative understanding of transporter levels in various cell types and tissues and new methods to modulate transporter function, such as allosteric modulators and targeted protein degraders of transporters. The section ends with a review of the effect of the gut microbiome on modulation of transporter function followed by a presentation of 3D cell cultures, which may enable in vivo predictions of transporter function. In the clinical section, we describe new genomic and pharmacogenomic research, highlighting important polymorphisms in transporters that are clinically relevant to many drugs. Finally, we describe new clinical tools, which are becoming increasingly available to enable precision medicine, with the application of tissue-derived small extracellular vesicles and real-world biomarkers.

P-glycoprotein (P-gp) plays a critical role in drug oral bioavailability, and modulation of this transporter can alter the safety and/or efficacy profile of substrate drugs. Individual oral molecular excipients that inhibit P-gp function have been considered a mechanism for improving drug absorption, but a systematic evaluation of the interaction of excipients with P-gp is critical for informed selection of optimal formulations of proprietary and generic drug products. A library of 123 oral molecular excipients was screened for their ability to inhibit P-gp in two orthogonal cell-based assays. β-Cyclodextrin and light green SF yellowish were identified as modest inhibitors of P-gp with IC50 values of 168 μM (95% CI, 118-251 μM) and 204 μM (95% CI, 5.9-1745 μM), respectively. The lack of effect of most of the tested excipients on P-gp transport provides a wide selection of excipients for inclusion in oral formulations with minimal risk of influencing the oral bioavailability of P-gp substrates.

The missense variant, breast cancer resistance protein (BCRP) p.Q141K, which encodes a reduced function BCRP, has been linked to poor response to allopurinol. Using a multifaceted approach, we aimed to characterize the relationship(s) between BCRP p.Q141K, the pharmacokinetics (PK) and pharmacodynamics (PD) of oxypurinol (the active metabolite of allopurinol), and serum uric acid (SUA) levels. A prospective clinical study (NCT02956278) was conducted in which healthy volunteers were given a single oral dose of 300 mg allopurinol followed by intensive blood sampling. Data were analyzed using noncompartmental analysis and population PK/PD modeling. Additionally, electronic health records were analyzed to investigate whether clinical inhibitors of BCRP phenocopied the effects of the p.Q141K variant with respect to SUA. Subjects homozygous for p.Q141K had a longer half-life (34.2 ± 12.2 h vs. 19.1 ± 1.42 h) of oxypurinol. The PK/PD model showed that women had a 24.8% lower volume of distribution. Baseline SUA was affected by p.Q141K genotype and renal function; that is, it changed by 48.8% for every 1 mg/dl difference in serum creatinine. Real-world data analyses showed that patients prescribed clinical inhibitors of BCRP have higher SUA levels than those that have not been prescribed inhibitors of BCRP, consistent with the idea that BCRP inhibitors phenocopy the effects of p.Q141K on uric acid levels. This study identified important covariates of oxypurinol PK/PD that could affect its efficacy for the treatment of gout as well as a potential side effect of BCRP inhibitors on increasing uric acid levels, which has not been described previously.

Numerous drugs are currently under accelerated clinical investigation for the treatment of coronavirus disease 2019 (COVID-19); however, well-established safety and efficacy data for these drugs are limited. The goal of this study was to predict the potential of 25 small molecule drugs in clinical trials for COVID-19 to cause clinically relevant drug-drug interactions (DDIs), which could lead to potential adverse drug reactions (ADRs) with the use of concomitant medications. We focused on 11 transporters, which are targets for DDIs. In vitro potency studies in membrane vesicles or HEK293 cells expressing the transporters coupled with DDI risk assessment methods revealed that 20 of the 25 drugs met the criteria from regulatory authorities to trigger consideration of a DDI clinical trial. Analyses of real-world data from electronic health records, including a database representing nearly 120,000 patients with COVID-19, were consistent with several of the drugs causing transporter-mediated DDIs (e.g., sildenafil, chloroquine, and hydroxychloroquine). This study suggests that patients with COVID-19, who are often older and on various concomitant medications, should be carefully monitored for ADRs. Future clinical studies are needed to determine whether the drugs that are predicted to inhibit transporters at clinically relevant concentrations, actually result in DDIs.

Human OAT1 and OAT3 play major roles in renal drug elimination and drug-drug interactions. However, there is little information on the interactions of drug metabolites with transporters. The goal of this study was to characterize the interactions of drug metabolites with OAT1 and OAT3 and compare their potencies of inhibition with those of their corresponding parent drugs. Using HEK293 cells stably transfected with OAT1 and OAT3, 25 drug metabolites and their corresponding parent drugs were screened for inhibitory effects on OAT1-and OAT3-mediated 6-carboxyfluorescein uptake at a screening concentration of 200 μM for all but 3 compounds. 20 and 24 drug metabolites were identified as inhibitors (inhibition > 50%) of OAT1 and OAT3, respectively. Seven drug metabolites were potent inhibitors of either or both OAT1 and OAT3 with Ki values less than 1 μM. 22 metabolites were more potent inhibitors of OAT3 than OAT1. Importantly, one drug and four metabolites were predicted to inhibit OAT3 at unbound plasma concentrations achieved clinically (Cmax,u/Ki values ≥ 0.1). In conclusion, our study highlights the potential interactions of drug metabolites with OAT1 and OAT3 at clinically relevant concentrations, suggesting that drug metabolites may modulate therapeutic and adverse drug response by inhibiting renal drug transporters.

The human solute carrier 22A (SLC22A) family consists of 23 members, representing one of the largest families in the human SLC superfamily. Despite their pharmacological and physiological importance in the absorption and disposition of a range of solutes, eight SLC22A family members remain classified as orphans. In this study, we used a multifaceted approach to identify ligands of orphan SLC22A15. Ligands of SLC22A15 were proposed based on phylogenetic analysis and comparative modeling. The putative ligands were then confirmed by metabolomic screening and uptake assays in SLC22A15 transfected HEK293 cells. Metabolomic studies and transporter assays revealed that SLC22A15 prefers zwitterionic compounds over cations and anions. We identified eight zwitterions, including ergothioneine, carnitine, carnosine, gabapentin, as well as four cations, including MPP+ , thiamine, and cimetidine, as substrates of SLC22A15. Carnosine was a specific substrate of SLC22A15 among the transporters in the SLC22A family. SLC22A15 transport of several substrates was sodium-dependent and exhibited a higher Km for ergothioneine, carnitine, and carnosine compared to previously identified transporters for these ligands. This is the first study to characterize the function of SLC22A15. Our studies demonstrate that SLC22A15 may play an important role in determining the systemic and tissue levels of ergothioneine, carnosine, and other zwitterions.

Requiring regional or in-country confirmatory clinical trials before approval of drugs already approved elsewhere delays access to medicines in low- and middle-income countries and raises drug costs. Here, we discuss the scientific and technological advances that may reduce the need for in-country or in-region clinical trials for drugs approved in other countries and limitations of these advances that could necessitate in-region clinical studies.

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

Food and drug products contain diverse and abundant small-molecule additives (excipients) with unclear impacts on human physiology, drug safety, and response. Here, we evaluate their potential impact on intestinal drug absorption. By screening 136 unique compounds for inhibition of the key intestinal transporter OATP2B1 we identified and validated 24 potent OATP2B1 inhibitors, characterized by higher molecular weight and hydrophobicity compared to poor or noninhibitors. OATP2B1 inhibitors were also enriched for dyes, including 8 azo (R-N=N-R') dyes. Pharmacokinetic studies in mice confirmed that FD&C Red No. 40, a common azo dye excipient and a potent inhibitor of OATP2B1, decreased the plasma level of the OATP2B1 substrate fexofenadine, suggesting that FD&C Red No. 40 has the potential to block drug absorption through OATP2B1 inhibition in vivo. However, the gut microbiomes of multiple unrelated healthy individuals as well as diverse human gut bacterial isolates were capable of inactivating the identified azo dye excipients, producing metabolites that no longer inhibit OATP2B1 transport. These results support a beneficial role for the microbiome in limiting the unintended effects of food and drug additives in the intestine and provide a framework for the data-driven selection of excipients. Furthermore, the ubiquity and genetic diversity of gut bacterial azoreductases coupled to experiments in conventionally raised and gnotobiotic mice suggest that variations in gut microbial community structure may be less important to consider relative to the high concentrations of azo dyes in food products, which have the potential to saturate gut bacterial enzymatic activity.

BACKGROUND: Radiation induces rapid bone loss and enhances bone resorption and adipogenesis, leading to an increased risk of bone fracture. There is still a lack of effective preventive or therapeutic method for irradiation-induced bone injury. Receptor activator of nuclear factor κB ligand (RANKL) provides the crucial signal to induce osteoclast differentiation and plays an important role in bone resorption. However, the mechanisms of radiation-induced osteoporosis are not fully understood. AIM: To investigate the role of CR6-interacting factor-1 (Crif1) in osteoclastogenesis after radiation and its possible mechanism. METHODS: C57BL/6 mice were exposed to Co-60 gamma rays and received 5 Gy of whole-body sublethal irradiation at a rate of 0.69 Gy/min. For in vitro study, mouse bone marrow mesenchymal stem/stromal cells (BM-MSCs) were irradiated with Co-60 at a single dose of 9 Gy. For osteoclast induction, monocyte-macrophage RAW264.7 cells were cocultured with mouse BM-MSCs for 7 d. ClusPro and InterProSurf were used to investigate the interaction interface in Crif1 and protein kinase cyclic adenosine monophosphate (cAMP)-activited catalytic subunit alpha complex. Virtual screening using 462608 compounds from the Life Chemicals database around His120 of Crif1 was carried out using the program Autodock_vina. A tetrazolium salt (WST-8) assay was carried out to study the toxicity of compounds to different cells, including human BM-MSCs, mouse BM-MSCs, and Vero cells. RESULTS: Crif1 expression increased in bone marrow cells after radiation in mice. Overexpression of Crif1 in mouse BM-MSCs and radiation exposure could increase RANKL secretion and promote osteoclastogenesis in vitro. Deletion of Crif1 in BM-MSCs could reduce both adipogenesis and RANKL expression, resulting in the inhibition of osteoclastogenesis. Deletion of Crif1 in RAW264.7 cells did not affect the receptor activator of nuclear factor κB expression or osteoclast differentiation. Following treatment with protein kinase A (PKA) agonist (forskolin) and inhibitor (H-89) in mouse BM-MSCs, Crif1 induced RANKL secretion via the cAMP/PKA pathway. Moreover, we identified the Crif1-protein kinase cyclic adenosine monophosphate-activited catalytic subunit alpha interaction interface by in silico studies and shortlisted interface inhibitors through virtual screening on Crif1. Five compounds dramatically suppressed RANKL secretion and adipogenesis by inhibiting the cAMP/PKA pathway. CONCLUSION: Crif1 promotes RANKL expression via the cAMP/PKA pathway, which induces osteoclastogenesis by binding to receptor activator of nuclear factor κB on monocytes-macrophages in the mouse model. These results suggest a role for Crif1 in modulating osteoclastogenesis and provide insights into potential therapeutic strategies targeting the balance between osteogenesis and adipogenesis for radiation-induced bone injury.

Mechanistic-understanding-based selection of excipients may improve formulation development strategies for generic drug products and potentially accelerate their approval. Our study aimed at investigating the effects of molecular excipients present in orally administered FDA-approved drug products on the intestinal efflux transporter, BCRP (ABCG2), which plays a critical role in drug absorption with potential implications on drug safety and efficacy. We determined the interactions of 136 oral molecular excipients with BCRP in isolated membrane vesicles and identified 26 excipients as BCRP inhibitors with IC50 values less than 5 μM using 3H-cholecystokinin octapeptide (3H-CCK8). These BCRP inhibitors belonged to three functional categories of excipients: dyes, surfactants, and flavoring agents. Compared with noninhibitors, BCRP inhibitors had significantly higher molecular weights and SLogP values. The inhibitory effects of excipients identified in membrane vesicles were also evaluated in BCRP-overexpressing HEK293 cells at similar concentrations. Only 1 of the 26 inhibitors of BCRP identified in vesicles inhibited BCRP-mediated 3H-oxypurinol uptake by more than 50%, consistent with the notion that BCRP inhibition depends on transmembrane or intracellular availability of the inhibitors. Collectively, the results of this study provide new information on excipient selection during the development of drug products with active pharmaceutical ingredients that are BCRP substrates.

BackgroundGlucagon-like peptide-1 (GLP-1) has been reported to have beneficial impacts on improving human's metabolism and ameliorating insulin resistance. While insulin is another important and conventional drug in diabetes treatment, but it has an adverse effect on weight gain.PurposeTo make sure whether GLP-1 and insulin play different roles in human adipose-derived stem cells (hADSCs).MethodsWe examined the in vitro roles and molecular mechanisms of liraglutide, a GLP-1 analogue, and human insulin on hADSCs isolated from subcutaneous adipose tissue. Different concentrations (0, 0.1, 1, 10, 100nM) of liraglutide and insulin were added to proliferation and differentiation medium of hADSCs, respectively.ResultsLiraglutide inhibits while insulin promotes the proliferation and differentiation at the concentration of 100nM. Moreover, the levels of GSK-3 increase during differentiation and liraglutide could down-regulate it when compared with insulin. We also find that the activation of phosphorylated GSK-3α and GSK-3β is involved in the differentiation roles. And classical and non-classical Wnt pathways all play roles in the differentiation, which are characterized with the up/down-regulation of the expression of adipogenesis genes such as PPAR-γ and CEBP-α.ConclusionLiraglutide and insulin have contrary effects on the proliferation and adipogenesis via Wnt pathway in primary cultured ADSCs. Those effects could partly explain the different roles of GLP-1 and insulin on weight gain and insulin resistance.

Antitumour treatments are evolving, including bacteria-mediated cancer therapy which is concurrently an ancient and cutting-edge approach. Salmonella typhimurium is a widely studied bacterial species that colonizes tumor tissues, showing oncolytic and immune system-regulating properties. It can be used as a delivery vector for genes and drugs, supporting conventional treatments that lack tumor-targeting abilities. This article summarizes recent evidence on the anticancer mechanisms of S. typhimurium alone and in combination with other anticancer treatments, suggesting that it may be a suitable approach to disease management.