A holistic strategy for quality and safety control of traditional Chinese medicines by the “iVarious” standard system

An effective quality control system is the key to ensuring the quality, safety and efficacy of traditional Chinese medicines(TCMs). However, the current quality standard research lacks top-level design and systematic design,mostly based on specific technologies or evaluation methods. To resolve the challenges and questions of quality control of TCMs, a brand-new quality standard system, named "iVarious", was proposed. The system comprises eight elements in a modular format. Meaning of every element was specifically illustrated via corresponding research instances. Furthermore, frankincense study was taken as an example for demonstrating standards and research process, based on the "i Various" system. This system highlighted a holistic strategy for effectiveness,security, integrity and systematization of quality and safety control standards of TCMs. The establishment of"i Various" integrates multi-disciplinary technologies and progressive methods, basis elements and key points of standard construction. The system provides a novel idea and technological demonstration for regulation establishment of TCMs quality standards.

Quality Control and Nonclinical Research on CAR-T Cell Products: General Principles and Key Issues

Adoptive cell therapy using chimeric antigen receptor T (CAR-T) cells, which is a promising cancer immunotherapy strategy, has been developing very rapidly in recent years. CAR-T cells are genetically modified T cells that can specifically recognize tumor specific antigens on the surface of tumor cells, and then effectively kill tumor cells. At present, exciting results are being achieved in clinical applications of CAR-T cells for patients with hematological malignancies. The research and development of CAR-T cells for various targets and for the treatment of solid tumors have become a hot topic worldwide, so an increasing number of investigational new drug applications (INDAs) and new drug applications (NDAs) of CAR-T cell products are expected to be submitted in future. The quality control and nonclinical research of these products are of great significance in ensuring the safety and effectiveness of these products;however, they also present great challenges and difficulties. This article discusses the general principles of and key issues regarding the quality control and nonclinical research of CAR-T cell products based on their product characteristics and on relevant guidelines for gene and cell therapy products.

Overexpression of wild-type HRAS drives non-alcoholic steatohepatitis to hepatocellular carcinoma in mice

Hepatocellular carcinoma(HCC),a prevalent solid carcinoma of significant concern,is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes.The etiology and pathological progression of non-alcoholic steatohepatitis(NASH)-related HCC is multifactorial and multistage.However,no single animal model can accurately mimic the full NASH-related HCC pathological progression,posing considerable challenges to transition and mechanistic studies.Herein,a novel conditional inducible wild-type human HRAS overexpressed mouse model(HRAS-HCC)was established,demonstrating 100%morbidity and mortality within approximately one month under normal dietary and lifestyle conditions.Advanced symptoms of HCC such as ascites,thrombus,internal hemorrhage,jaundice,and lung metastasis were successfully replicated in mice.In-depth pathological features of NASH-related HCC were demonstrated by pathological staining,biochemical analyses,and typical marker gene detections.Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival,further confirming the accuracy and reliability of the model.Based on protein-protein interaction(PPI)network and RNA sequencing analyses,we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis,with subsequent progression to HCC.Collectively,our study successfully duplicated natural sequential progression in a single murine model over a very short period,providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.

Bio-soft matter derived from traditional Chinese medicine:Characterizations of hierarchical structure,assembly mechanism,and beyond

Structural and functional explorations on bio-soft matter such as micelles,vesicles,nanoparticles,aggregates or polymers derived from traditional Chinese medicine(TCM)has emerged as a new topic in the field of TCM.The discovery of such cross-scaled bio-soft matter may provide a unique perspective for unraveling the new effective material basis of TCM as well as developing innovative medicine and biomaterials.Despite the rapid rise of TCM-derived bio-soft matter,their hierarchical structure and assembly mechanism must be unambiguously probed for a further in-depth understanding of their pharmacological activity.In this review,the current emerged TCM-derived bio-soft matter assembled from either small molecules or macromolecules is introduced,and particularly the unambiguous elucidation of their hierarchical structure and assembly mechanism with combined electron microscopic and spectroscopic techniques is depicted.The pros and cons of each technique are also discussed.The future challenges and perspective of TCM-derived bio-soft matter are outlined,particularly the requirement for their precise in situ structural determination is highlighted.

Insights into Euphorbia diversity: Probing the contrasts between Euphorbia fischeriana Steud and Euphorbia ebracteolata Hayata

In traditional Chinese medicine(TCM),Euphorbia fischeriana Steud(E.fischeriana)and Euphorbia ebracteolata Hayata(E.ebracteolata),commonly referred to as“Langdu”,are widely extensively utilized for treating lymphatic tuberculosis and ringworm[1].Both plant species are perennial herbaceous plants mainly distributed in northeastern China,Mongolia,Russia(Siberia),and Republic of Korea[2].There have been many reports on the chemical constituents and pharmacological effects of the two plant species,which has made more and more researchers realize that there may be differences between E.fischeriana and E.ebracteolata.In some cases,long-term improper use of herbal medicines can even lead to life-threatening conditions[3,4].Therefore,it is essential to employ an effective technology to differentiate between these two plants based on their chemical constituents and biological activities,so as to reduce the harm caused by the mixing and misuse of medicinal materials.Therefore,the present paper describes a study of the differences between E.ebracteolata and E.fischeriana,using untargeted plant metabolomics and biological activity evaluations.This study aims to provide valuable insight into their equivalence and potential interchangeability in TCM and clinical medication.

LAL test and RPT for endotoxin detection of CPT-11/DSPE-mPEG_(2000) nanoformulation: What if traditional methods are not applicable?

Endotoxin detection is an important step in drug characterization. Herein we found that a chemotherapeutic drug nanoformulation composed of irinotecan hydrochloride(CPT-11) and an amphiphilic molecule DSPE-mPEG_(2000) can interfere with the limulus amebocyte lysate assay(LAL). Furthermore, the rabbit pyrogen test(RPT) results indicated that at a relatively high dosage, the drug irinotecan hydrochloride can induce a hypothermia effect which may render the RPT results ambiguous in determination of the safety of the drug formulation.Our findings demonstrate limitations of endotoxin detection in micellar drugs,and call for the necessity of developing reliable endotoxin detection methods that can overcome the interference of nanomaterials in order to better ensure the drug safety of patients in future pharmaceutical drug development.

SpectraTr:A novel deep learning model for qualitative analysis of drug spectroscopy based on transformer structure

The drug supervision methods based on near-infrared spectroscopy analysis are heavily dependent on the chemometrics model which characterizes the relationship between spectral data and drug categories.The preliminary application of convolution neural network in spectral analysis demonstrates excellent end-to-end prediction ability,but it is sensitive to the hyper-parameters of the network.The transformer is a deep-learning model based on self-attention mechanism that compares convolutional neural networks(CNNs)in predictive performance and has an easy-todesign model structure.Hence,a novel calibration model named SpectraTr,based on the transformer structure,is proposed and used for the qualitative analysis of drug spectrum.The experimental results of seven classes of drug and 18 classes of drug show that the proposed SpectraTr model can automatically extract features from a huge number of spectra,is not dependent on pre-processing algorithms,and is insensitive to model hyperparameters.When the ratio of the training set to test set is 8:2,the prediction accuracy of the SpectraTr model reaches 100%and 99.52%,respectively,which outperforms PLS DA,SVM,SAE,and CNN.The model is also tested on a public drug data set,and achieved classification accuracy of 96.97%without preprocessing algorithm,which is 34.85%,28.28%,5.05%,and 2.73%higher than PLS DA,SVM,SAE,and CNN,respectively.The research shows that the SpectraTr model performs exceptionally well in spectral analysis and is expected to be a novel deep calibration model after Autoencoder networks(AEs)and CNN.

Worm-like micelles facilitate the intestinal mucus diffusion and drug accumulation for enhancing colorectal cancer therapy

Local delivery of nanoparticles holds promise for colorectal cancer(CRC)therapy.However,the presence of the mucus layer on the epithelium poses a significant challenge to drug delivery,thereby adversely affecting treatment efficiency.It is crucial to develop efficient drug delivery carriers that can effectively overcome mucus barriers to treat colorectal cancer.Herein,we utilized poly(1,4-butadiene)-b-poly(ethylene oxide)polymers to prepare four distinct geometries of polymeric micelles,namely linear micelles(LMs),worm-like micelles(WLMs),large spherical micelles(LSMs),and small spherical micelles(SSMs)to investigate the influence of shape effects on overcoming colonic mucosal barrier.We found that the carriers exhibited diverse shapes while maintaining comparable physicochemical properties.Of these,WLMs had an aspect ratio similar to segmented filamentous bacteria,which exhibited superior mucus penetration ability,leading to prolonged drug release kinetics and faster entry into epithelial cells compared to LSMs.Furthermore,rectally administrated 10-hydroxycamptothecin-loaded WLMs traversed the colorectal mucus in orthotopic CRC nude mice model,penetrated and accumulated within tumor tissue,and effectively aggregated within cancer cells,thereby inducing significantly robust antitumor outcomes in vivo.These findings underscore the significance of shape design in overcoming colonic mucosal absorption barriers,offering a novel approach for the development of drug delivery carriers tailored for effective tumor therapy.

Anti-inflammatory mechanism of the non-volatile ingredients performance liquid chromatography-heated electron spray ionization-high resolution mass spectroscope and cell metabolomics

OBJECTIVE:To explore the anti-inflammatory components and mechanism of the non-volatile ingredients of patchouli.METHODS:High performance liquid chromatographyheated electron spray ionization-high resolution mass spectroscope(HPLC-HESI-HRMS)was used to analyze the chemical constituents of the non-volatile ingredients of patchouli.The anti-inflammatory activity of ingredients was evaluated using lipopolysaccharide(LPS)induced RAW264.7 cell inflammation model,and the antiinflammatory mechanism was investigated using multivariate statistical analysis of cell metabolomics.RESULTS:The non-volatile ingredients of patchouli were characterized by HPLC-HESI-HRMS,and 36 flavonoids and 18 other components were identified.These ingredients of patchouli not only had a good protective effect on the LPS-induced inflammation model of RAW264.7 cells,but also regulated the expression levels of arginine,L-leucine,cholesterol,fructose and sorbitol by down-regulating arginine metabolism,aminoacyl-tRNA biosynthesis,polyol/sorbitol pathway,so as to reduce inflammation and reduce cell damage.CONCLUSION:The non-volatile ingredients of patchouli had good anti-inflammatory effect and exerted its curative effect by regulating endogenous metabolic pathway to reduce inflammatory response.

Mortality outcomes of low-dose computed tomography screening for lung cancer in urban China:a decision analysis and implications for practice

Background: Mortality outcomes in trials of low-dose computed tomography(CT) screening for lung cancer are inconsistent. This study aimed to evaluate whether CT screening in urban areas of China could reduce lung cancer mortality and to investigate the factors that associate with the screening effect.Methods: A decision tree model with three scenarios(low-dose CT screening, chest X-ray screening, and no screening) was developed to compare screening results in a simulated Chinese urban cohort(100,000 smokers aged45-80 years). Data of participant characteristics were obtained from national registries and epidemiological surveys for estimating lung cancer prevalence. The selection of other tree variables such as sensitivities and specificities of low-dose CT and chest X-ray screening were based on literature research. Differences in lung cancer mortality(primary outcome), false diagnoses, and deaths due to false diagnosis were calculated. Sensitivity analyses were performed to identify the factors that associate with the screening results and to ascertain worst and optimal screening effects considering possible ranges of the variables.Results: Among the 100,000 subjects, there were 448,541, and 591 lung cancer deaths in the low-dose CT, chest X-ray, and no screening scenarios, respectively(17.2% reduction in low-dose CT screening over chest X-ray screening and 24.2% over no screening). The costs of the two screening scenarios were 9387 and 2497 false diagnoses and 7and 2 deaths due to false diagnosis among the 100,000 persons, respectively. The factors that most influenced death reduction with low-dose CT screening over no screening were lung cancer prevalence in the screened cohort, lowdose CT sensitivity, and proportion of early-stage cancers among low-dose CT detected lung cancers. Considering all possibilities, reduction in deaths(relative numbers) with low-dose CT screening in the worst and optimal cases were16(5.4%) and 288(40.2%) over no screening, respectively.Conclusions: In terms of mortality outcomes, our findings favor conducting low-dose CT screening in urban China.However, approaches to reducing false diagnoses and optimizing important screening conditions such as enrollment criteria for screening are highly needed.

A rapid reporter assay for recombinant human brain natriuretic peptide(rhBNP)by GloSensor technology

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide （rhBNP）. In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGIo-G1 was constructed by transfecting pGloSensorTM 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGIo-G1 showed high precision with intraassay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R^2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay （paired t test,p = 0.630）. All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP.

Safety and immunogenicity of human papillomavirus-16/18 AS04-adjuvanted vaccine in healthy Chinese females aged 15 to 45 years:a phase Ⅰ trial

Globally,about 70% of cervical cancers are associated with human papillomavirus (HPV)-16 or HPV-18 infection.A meta-analysis of epidemiologic studies in China showed that HPV was present in 98% of cervical cancer samples.The HPV-16/18 AS04-adjuvanted vaccine Cervarix has shown a high level of protection against HPV-16/18 infections and associated cervical lesions.This phase Ⅰ trial (NCT00549900) assessed the safety,tolerability,and immunogenicity of the vaccine in Chinese.Thirty healthy Chinese females,aged 15 to 45 years with a median age of 29.5 years,received three doses of Cervarix in Months 0,1,and 6.Safety was assessed via recording solicited local and systemic symptoms within 7 days and unsolicited symptoms within 30 days after each vaccination.Serious adverse events,new onset of chronic diseases,and other medically significant conditions were recorded throughout this trial.As an exploratory objective,HPV-16/18 antibody titers were determined by enzyme-linked immunosorbent assay in serum samples collected in Months 0 and 7.Pain at the injection site was the most frequently reported local symptom.Two subjects reported medically significant adverse events.Both cases were assessed as unrelated to vaccination by the investigator.In Month 7,100% seroconversion was observed for both anti-HPV-16 and anti-HPV-18 with high geometric mean antibody titers.HPV-16/18 AS04-adjuvanted vaccine,evaluated for the first time in Chinese females,was generally well tolerated and immunogenic,as previously shown in global studies.

Multi-manufacturer drug identification based on near infrared spectroscopy and deep transfer learning

Near infrared(NIR)spectrum analysis technology has outstanding advantages such as rapid,nondestructive,pollution-free,and is widely used in food,pharmaceutical,petrochemical,agricultural products production and testing industries.Convolutional neural network(CNN)is one of the most successful methods in big data analysis because of its powerful feature ex-traction and abstraction ability,and it is especially suitable for solving multi-classification problems.CNN-based transfer learning is a machine learning technique,which migrates para-meters of trained model to the new one to improve the performance.The transfer learning strategy can speed up the learning efficiency of the model instead of learning from scratch.In view of the difficulty in acquisition of drug NIR spectral data and high labeling cost,this paper proposes three simple but very effective transfer learning methods for multi-manufacturer identification of drugs based on one-dimensional CNN.Compared with the original CNN,the transfer learning method can achieve better classification performance with fewer NIR spectral data,which greatly reduces the dependence on labeled NIR spectral data.At the same time,this paper also compares and discusses three different transfer learning methods,and selects the most suitable transfer learning model for drug NIR spectral data analysis.Compared with the current popular methods,such as SVM,BP,AE and ELM,the proposed method achieves higher classification accuracy and scalability in multi-variety and multi-manufacturer NIR spectrum classification experiments.

Multi-dimensional visualization for the morphology of lubricant stearic acid particles and their distribution in tablets

The shapes of particles and their distribution in tablets, controlled by pretreatment and tableting process, determine the pharmaceutical performance of excipient like lubricant. This study aims to provide deeper insights to the relationship of the morphology and spatial distribution of stearic acid(SA) with the lubrication efficiency, as well as the resulting tablet property. Unmodified SA particles as flat sheet-like particles were firstly reprocessed by emulsification in hot water to obtain the reprocessed SA particles with spherical morphology. The three-dimensional(3 D) information of SA particles in tablets was detected by a quantitative and non-invasive 3 D structure elucidation technique, namely, synchrotron radiation X-ray micro-computed tomography(SR-μCT). SA particles in glipizide tablets prepared by using unmodified SA(GUT), reprocessed SA(GRT), as well as reference listed drug(RLD) of glipizide tablets were analyzed by SR-μCT. The results showed that the reprocessed SA with better flowability contributed to similarity of breaking forces between that of GRT and RLD. SA particles in GRT were very similar to those in RLD with uniform morphology and particle size, while SA particles in GUT were not evenly distributed. These findings not only demonstrated the feasibility of SR-μCT as a new method in revealing the morphology and spatial distribution of excipient in drug delivery system, but also deepened insights of solid dosage form design into a new scale by powder engineering.

Blends of hydrophobic and swelling agents in the swelling layer in the preparation of delayed-release pellets of a hydrophilic drug with low MW: Physicochemical characterizations and in-vivo evaluations

In this study,a hydrophobic material,ethylcellulose,which was used as its aqueous suspension Surelease^(®),was combined with a swelling agent as the swelling layer to prepare delayed-release pellets for Danshensu,which is a hydrophilic drug with low MW.A rupturable,delayed-release pellet consists of a drug core,a swelling layer containing a swelling agent(cross-linked sodium carboxymethyl cellulose)with a hydrophobic agent(Surelease^(®)),and a controlled layer composed by an insoluble,water-permeable polymeric coating(aqueous ethylcellulose dispersions)was developed in a fluidised bed.Results showed that blending Surelease^(®)into the swelling layer could effectively extend the release of Danshensu from the pellets,which may be attributed to the slowed swelling rate by reduction of water penetration and improvement of mechanical integrity of the swelling layer.Drug in the delayed pellets showed sustained release in beagle dogs after oral administration with comparable in-vivo exposure to the uncoated drug pellets.In conclusion,blends of hydrophobic and swelling agents in the swelling layer in doublemembrane pellets could achieve a delayed drug-release profile in vitro,as well as delayed and sustained absorption in vivo for highly soluble,low-MW drug.The present study highlighted the potential use of a delayed-release system for other hydrophilic,low-MW drugs to meet the formulation requirements for chronopharmacological diseases.

Examine the Correlation between Heat Shock Protein IbpA and Heat Tolerance in Cronobacter sakazakii

We used a proteomic approach to identify IbpA in Cronobacter sakazokii （C. sakazaki）, which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 AibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 AibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coil O157：H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.

Development and implementation of incident response near-infrared models for analyzing contaminated medicines containing diethylene glycol solvent

Samples of preparations contaminated by diethylene glycol(DEG),diethylene glycol raw materials and laboratory prepared solutions were measured to get NIR spectra.Then the iden-tification models were developed using the collected spectra and the spectra of distilled water,propylene glyool and the preparations without diethylene glyool.Besides,the quantification model was also established for determining the concentration of diethylene glyool in the pre-parations.V alidation results show the identification and quantification models have ideal pre-diction performance.The emergency NIR models are rapid,easy to use and accurate,and can be implemented for identifying diethylene glycol raw material,screening the preparations contam-inated by diethylene glycol in the markets and analyzing the concentrations of DEG.

Progress and Challenges of Reference Standard and Its New Form: Digital Reference Standard

Number and quantities of reference standards (RS) needed for the quality control of drugs are increasing, bringing great pressure to the calibration and using company. This manuscript summarized the four generations for the development of RS including physical RS, paper atlas, substitute RS and electronic databases. The advantages and disadvantages of each generation were summarized. The concept of digital RS (DRS) was proposed based on this, and summed up the definition, advantages, and technical architecture of DRS. The 10 characteristics of five aspects of the DRS were discussed including digital, multi-dimension, big data, cloud computing, internet, internet of things, sharing, multi-terminal, intelligence, and compliance certification. Then, the necessity of its formation and application in the medicine holistic quality control of internet plus era was discussed in this manuscript.

The interaction between polyphyllin I and SQLE protein induces hepatotoxicity through SREBP-2/HMGCR/SQLE/LSS pathway

Polyphyllin I(PPI)and polyphyllin II(PII)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPI and PII exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepatotoxicity of PPI and PII was associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the cholesterol synthesis,selected as the potential targets,were confirmed by the molecular docking,the overexpression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPI could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPI-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon a2b

Recombinant human interferon a2b(rhIFNa2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNa2b is complex.In this study,an anti-rhIFNa2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNa2b.RhIFNa2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNa2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNa2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNa2b detection assay had a limit of detection of 1 mg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other commercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.