Significance and challenges of stereoselectivity assessing methods in drug metabolism

Stereoselectivity in drug metabolism can not only influence the pharmacological activities, tolerability,safety, and bioavailability of drugs directly, but also cause different kinds of drug–drug interactions. Thus,assessing stereoselectivity in drug metabolism is of great significance for pharmaceutical research and development(R&D) and rational use in clinic. Although there are various methods available for assessing stereoselectivity in drug metabolism, many of them have shortcomings. The indirect method of chromatographic methods can only be applicable to specific samples with functional groups to be derivatized or form complex with a chiral selector, while the direct method achieved by chiral stationary phases(CSPs) is expensive. As a detector of chromatographic methods, mass spectrometry(MS) is highly sensitive and specific, whereas the matrix interference is still a challenge to overcome. In addition, the use of nuclear magnetic resonance(NMR) and immunoassay in chiral analysis are worth noting. This review presents several typical examples of drug stereoselective metabolism and provides a literature-based evaluation on current chiral analytical techniques to show the significance and challenges of stereoselectivity assessing methods in drug metabolism.

Potential use of a dried saliva spot(DSS)in therapeutic drug monitoring and disease diagnosis

In recent years,scientific researchers have increasingly become interested in noninvasive sampling methods for therapeutic drug monitoring and disease diagnosis.As a result,dried saliva spot(DSS),which is a sampling technique for collecting dried saliva samples,has been widely used as an alternative matrix to serum for the detection of target molecules.Coupling the DSS method with a highly sensitive detection instrument improves the efficiency of the preparation and analysis of biological samples.Furthermore,dried blood spots,dried plasma spots,and dried matrix spots,which are similar to those of the DSS method,are discussed.Compared with alternative biological fluids used in dried spot methods,including serum,tears,urine,and plasma,saliva has the advantage of convenience in terms of sample collection from children or persons with disabilities.This review aims to provide integral strategies and guidelines for dried spot methods to analyze biological samples by illustrating several dried spot methods.Herein,we summarize recent advancements in DSS methods from June 2014 to March 2021 and discuss the advantages and disadvantages of the key aspects of this method,including sample preparation and method validation.Finally,we outline the challenges and prospects of such methods in practical applications.

Effects of polyol excipient stability during storage and use on the quality of biopharmaceutical formulations

Biopharmaceuticals are formulated using a variety of excipients to maintain their storage stability.However,some excipients are prone to degradation during repeated use and/or improper storage,and the impurities generated by their degradation are easily overlooked by end users and are usually not strictly monitored,affecting the stability of biopharmaceuticals.In this study,we evaluated the degradation profile of polyol excipient glycerol during repeated use and improper storage and identified an unprecedented cyclic ketal impurity using gas chromatography with mass spectrometry(GC-MS).The other polyol excipient,mannitol,was much more stable than glycerol.The effects of degraded glycerol and mannitol on the stability of the model biopharmaceutical pentapeptide,thymopentin,were also evaluated.The thymopentin content was only 66.4% in the thymopentin formulations with degraded glycerol,compared to 95.8% in other formulations after the stress test.Most glycerol impurities(i.e.,aldehydes and ketones)reacted with thymopentin,affecting the stability of thymopentin formulations.In conclusion,this work suggests that more attention should be paid to the quality changes of excipients during repeated use and storage.Additional testing of excipient stability under real or accelerated conditions by manufacturers would help avoid unexpected and painful results.

Preface for the special issue on analysis of drug or drug targets by molecular imaging

To increase the chance of a successful outcome in clinic and in the development of innovative drugs,researchers aim to provide more compre hensive information about the disease and drugs to adapt treatment decisions according to an individual disease's mo-lecular characteristics.It is thus increasingly desirable to illuminate fund amental molecular pathways of the drug and its targets inside organisms in a non-invasive manner.Technologies developed in molecular imaging assist in visualizing,characterizing,and quanti-fying targets of interest at the molecular level within intact living organisms.This special issue of Joumal of Pharmaceutical Analysis is therefore dedicated to highlighting current progresses made in molecular imaging towards various drugs,important or promising drug targets,and their interactions,as well as to providing a forum for sharing new methods reported recently for the efficient phar-maceutical analysis.

Recent advances and perspectives of nucleic acid detection for coronavirus

The recent pneumonia outbreak caused by a novel coronavirus(SARS-CoV-2)is posing a great threat to global public health.Therefore,rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments,saving people's lives and preventing epidemics.It is important to establish a quick standard diagnostic test for the detection of the infectious disease(COVID-19)to prevent subsequent secondary spread.Polymerase chain reaction(PCR)is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity.Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler opera-tion.A variety of improved or new approaches also have been developed.This review summarizes the currently available detection methods for coronavirus nucleic acid.It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coro-navirus infection.

Current developments of bioanalytical sample preparation techniques in pharmaceuticals

Sample preparation is considered as the bottleneck step in bioanalysis because each biological matrix has its own unique challenges and complexity.Competent sample preparation to extract the desired analytes and remove redundant components is a crucial step in each bioanalytical approach.The matrix effect is a key hurdle in bioanalytical sample preparation,which has gained extensive consideration.Novel sample preparation techniques have advantages over classical techniques in terms of accuracy,automation,ease of sample preparation,storage,and shipment and have become increasingly popular over the past decade.Our objective is to provide a broad outline of current developments in various bioanalytical sample preparation techniques in chromatographic and spectroscopic examinations.In addition,how these techniques have gained considerable attention over the past decade in bioanalytical research is mentioned with preferred examples.Modern trends in bioanalytical sample preparation techniques,including sorbent-based microextraction techniques,are primarily emphasized.

Recent advances in construction of small molecule-based fluorophoredrug conjugates

As a powerful tool to advance drug discovery,molecular imaging may provide new insights into the process of drug effect and therapy at cellular and molecular levels.When compared with other detection methods,fluorescence-based strategies are highly attractive and can be used to illuminate pathways of drugs’transport,with multi-color capacity,high specificity and good sensitivity.The conjugates of fluorescent molecules and therapeutic agents create exciting avenues for real-time monitoring of drug delivery and distribution,both in vitro and in vivo.In this short review,we discuss recent developments of small molecule-based fluorophore-drug conjugates,including non-cleavable and cleavable ones,that are capable of visualizing drug delivery.

Development of a UHPLC-MS/MS method for the quantification of ilaprazole enantiomers in rat plasma and its pharmacokinetic application

In Korea and China,ilaprazole is a widely used proton pump inhibitor in the treatment of gastric ulcers.In this study,a specific and sensitive LC-MS/MS method has been developed and validated for the quantification of ilaprazole enantiomers in the rat plasma,using R-lansoprazole as the internal standard.The enantioseparation was achieved on a CHIRALPAK AS-RH column(4.6 mm×150 mm,i.d.5 mm),with a mobile phase composed of 10 m M ammonium acetate aqueous solution and acetonitrile(60:40,V/V),at a flow-rate of 0.5 m L/min.The method was validated over the concentration range of 0.5 e300 ng/m L for both,R-and S-ilaprazole.The lower limit of quantification was 0.5 ng/m L for both enantiomers.The relative standard deviation(RSD)of intra-and inter-day precision of R-ilaprazole and S-ilaprazole was less than 10.9%,and the relative error accuracy(RE)ranged fromà0.5%e2.0%.Finally,the method was successfully evaluated in rats in a stereoselective pharmacokinetic study of the ilaprazole racemate.

Research advances in the detection of miRNA

MicroRNAs (miRNAs) are a family of endogenous, small (approximately 22 nucleotides in length), noncoding, functional RNAs. With the development of molecular biology, the research of miRNA biological function has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Traditional methods for miRNA detection do not meet current demands. In particular, nanomaterial-based methods, nucleic acid amplification-based methods such as rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA) and some enzyme-free amplifications have been employed widely for the highly sensitive detection of miRNA. MiRNA functional research and clinical diagnostics have been accelerated by these new techniques. Herein, we summarize and discuss the recent progress in the development of miRNA detection methods and new applications. This review will provide guidelines for the development of follow-up miRNA detection methods with high sensitivity and specificity, and applicability to disease diagnosis and therapy.

Recent progress in the molecular imaging of therapeutic monoclonal antibodies

Therapeutic monoclonal antibodies have become one of the central components of the healthcare system and continuous efforts are made to bring innovative antibody therapeutics to patients in need.It is equally critical to acquire sufficient knowledge of their molecular structure and biological functions to ensure the efficacy and safety by incorporating new detection approaches since new challenges like individual differences and resistance are presented.Conventional techniques for determining antibody disposition including plasma drug concentration measurements using LC-MS or ELISA,and tissue distribution using immunohistochemistry and immunofluorescence are now complemented with molecular imaging modalities like positron emission tomography and near-infrared fluorescence imaging to obtain more dynamic information,while methods for characterization of antibody’s interaction with the target antigen as well as visualization of its cellular and intercellular behavior are still under development.Recent progress in detecting therapeutic antibodies,in particular,the development of methods suitable for illustrating the molecular dynamics,is described here.

The metabolism and hepatotoxicity of ginkgolic acid(17:1) in vitro

Pharmacological activities and adverse side effects of ginkgolic acids(GAs), major components in extracts from the leaves and seed coats of Ginkgo biloba L, have been intensively studied. However, there are few reports on their hepatotoxicity. In the present study, the metabolism and hepatotoxicity of GA(17:1), one of the most abundant components of GAs, were investigated. Kinetic analysis indicated that human and rat liver microsomes shared similar metabolic characteristics of GA(17:1) in phase I and II metabolisms. The drug-metabolizing enzymes involved in GA(17:1) metabolism were human CYP1 A2, CYP3 A4, UGT1 A6, UGT1 A9, and UGT2 B15, which were confirmed with an inhibition study of human liver microsomes and recombinant enzymes. The MTT assays indicated that the cytotoxicity of GA(17:1) in HepG2 cells occurred in a time-and dose-dependent manner. Further investigation showed that GA(17:1) had less cytotoxicity in primary rat hepatocytes than in HepG2 cells and that the toxicity was enhanced through CYP1 A-and CYP3 A-mediated metabolism.

Upregulation of miR-489-3p and miR-630 inhibits oxaliplatin uptake in renal cell carcinoma by targeting OCT2

Renal cell carcinoma(RCC) is one of the most common malignant tumors affecting the urogenital system, accounting for 90% of renal malignancies. Traditional chemotherapy options are often the front-line choice of regimen in the treatment of patients with RCC, but responses may be modest or limited due to resistance of the tumor to anticarcinogen. Downregulated expression of organic cation transporter OCT2 is a possible mechanism underlying oxaliplatin resistance in RCC treatment. In this study, we observed that mi R-489-3 p and mi R-630 suppress OCT2 expression by directly binding to the OCT2 30-UTR. Meanwhile, via 786-O-OCT2-mi RNAs stable expression cell models, we found that mi RNAs could repress the classic substrate 1-methyl-4-phenylpyridinium(MPP+), fluorogenic substrate N,N-dimethyl-4-(2-pyridin-4-ylethenyl) aniline(ASP+), and oxaliplatin uptake by OCT2 both in vitro and in xenografts. In 33 clinical samples, mi R-489-3 p and mi R-630 were significantly upregulated in RCC, negatively correlating with the OCT2 expression level compared to that in adjacent normal tissues, using tissue microarray analysis and q PCR validation. The increased binding of c-Myc to the promoter of pri-mi R-630, responsible for the upregulation of mi R-630 in RCC, was further evidenced by chromatin immunoprecipitation and dual-luciferase reporter assay. Overall, this study indicated that mi R-489-3 p and mi R-630 function as oncotherapy-obstructing micro RNAs by directly targeting OCT2 in RCC.

TSNAdb v2.0:The Updated Version of Tumor-specific Neoantigen Database

In recent years,neoantigens have been recognized as ideal targets for tumor immunotherapy.With the development of neoantigen-based tumor immunotherapy,comprehensive neoantigen databases are urgently needed to meet the growing demand for clinical studies.We have built the tumor-specific neoantigen database(TSNAdb)previously,which has attracted much attention.In this study,we provide TSNAdb v2.0,an updated version of the TSNAdb.TSNAdb v2.0 offers several new features,including(1)adopting more stringent criteria for neoantigen identification,(2)providing predicted neoantigens derived from three types of somatic mutations,and(3)collecting experimentally validated neoantigens and dividing them according to the experimental level.

Mechanism for ginkgolic acid(15：1)-induced MDCK cell necrosis： Mitochondria and lysosomes damages and cell cycle arrest

Ginkgolic acids(GAs), primarily found in the leaves, nuts, and testa of ginkgo biloba, have been identified with suspected allergenic, genotoxic and cytotoxic properties. However, little information is available about GAs toxicity in kidneys and the underlying mechanism has not been thoroughly elucidated so far. Instead of GAs extract, the renal cytotoxicity of GA(15 : 1), which was isolated from the testa of Ginkgo biloba, was assessed in vitro by using MDCK cells. The action of GA(15 : 1) on cell viability was evaluated by the MTT and neutral red uptake assays. Compared with the control, the cytotoxicity of GA(15 : 1) on MDCK cells displayed a time-and dose-dependent manner, suggesting the cells mitochondria and lysosomes were damaged. It was confirmed that GA(15 : 1) resulted in the loss of cells mitochondrial trans-membrane potential(ΔΨm). In propidium iodide(PI) staining analysis, GA(15 : 1) induced cell cycle arrest at the G0/G1 and G2/M phases, influencing on the DNA synthesis and cell mitosis. Characteristics of necrotic cell death were observed in MDCK cells at the experimental conditions, as a result of DNA agarose gel electrophoresis and morphological observation of MDCK cells. In conclusion, these findings might provide useful information for a better understanding of the GA(15 : 1) induced renal toxicity.

Screening and verifying potential NTCP inhibitors from herbal medicinal ingredients using the LLC-PK1 cell model stably expressing human NTCP

NTCP is specifically expressed on the basolateral membrane of hepatocytes, participating in the enterohepatic circulation of bile salts, especially conjugated bile salts, to maintain bile salts homeostasis. In addition, recent studies have found that NTCP is a functional receptor of HBV and HDV. Therefore, it is important to study the interaction between drugs and NTCP and identify the inhibitors/substrates of NTCP. In the present study, a LLC-PK1 cell model stably expressing human NTCP was established, which was simple and suitable for high throughput screening, and utilized to screen and verify the potential inhibitors of NTCP from 102 herbal medicinal ingredients. The results showed that ginkgolic acid(GA)(13 : 0), GA(15 : 1), GA(17 : 1), erythrosine B, silibinin, and emodin have inhibitory effects on NTCP uptake of TCNa in a concentration-dependent manner. Among them, GA(13 : 0) and GA(15 : 1) exhibited the stronger inhibitory effects, with IC_(50) values being less than 8.3 and 13.5 mmol·L^(-1), respectively, than the classical inhibitor, cyclosporin A(CsA)(IC_(50) = 20.33 mmol·L^(-1)). Further research demonstrated that GA(13 : 0), GA(15 : 1), GA(17 : 1), silibinin, and emodin were not substrates of NTCP. These findings might contribute to a better understanding of the disposition of the herbal ingredients in vivo, especially in biliary excretion.

TSNAdb: A Database for Tumor-specific Neoantigens from Immunogenomics Data Analysis

Tumor-specific neoantigens have attracted much attention since they can be used as biomarkers to predict therapeutic effects of immune checkpoint blockade therapy and as potential targets for cancer immunotherapy. In this study, we developed a comprehensive tumor-specific neoantigen database(TSNAdb v1.0), based on pan-cancer immunogenomic analyses of somatic mutation data and human leukocyte antigen(HLA) allele information for 16 tumor types with 7748 tumor samples from The Cancer Genome Atlas(TCGA) and The Cancer Immunome Atlas(TCIA). We predicted binding affinities between mutant/wild-type peptides and HLA class I molecules by NetMHCpan v2.8/v4.0, and presented detailed information of 3,707,562/1,146,961 potential neoantigens generated by somatic mutations of all tumor samples. Moreover, we employed recurrent mutations in combination with highly frequent HLA alleles to predict potential shared neoantigens across tumor patients,which would facilitate the discovery of putative targets for neoantigen-based cancer immunotherapy.TSNAdb is freely available at http://biopharm.zju.edu.cn/tsnadb.

A multivalent biparatopic EGFR-targeting nanobody drug conjugate displays potent anticancer activity in solid tumor models

Dear Editor,The epidermal growth factor receptor(EGFR),one member of the ErbB family of receptor tyrosine kinases,has been implicated in various epithelial malignancies.As a clinically validated target,both small-molecule tyrosine kinase inhibitors and antibody-based drugs have been approved by regulatory agencies.Anti-EGFR antibodyci rug conjugates(ADCs)could kill target tumor cells irrespective of EGFR signaling.However,cancer patients treated with EGFR-targeted antibody therapy eventually develop acquired epitope substitutions such as S492R,G465R,G465E,and 1491M.

The inhibition mechanism of the uptake of lamivudine via human organic anion transporter 1 by Stellera chamaejasme L. extracts

Stellera chamaejasme L.is a traditional Chinese medicine with a long history to treat stubborn skin ulcer,and it also has antiviral and antitumor effects.Neochamaejasmine B(NCB),Neochamaejasmine A(NCA)and Chamaechromone(CMC)are the major components in dried roots of Stellera chamaejasme L..Our studies suggested that NCB,NCA and CMC are inhibitors of Organic anion transporter 1(OAT1).OAT1 is encoded by solute carrier family 22 member 6 gene(SLC22 A6)in humans and plays a critical role in the organic anion drug uptake and excretion in the kidney.Lamivudine is the typical substrate of OAT1 and is frequently used in combination with other antiviral drugs in clinical antiviral treatments.The aim of this study is to investigate the interaction and its mechanism between these bi-flavone components in Stellera chamaejasme L.and lamivudine via OAT1 both in vitro and in vivo.In vitro,the uptake studies in Madin-Darby canine kidney(MDCK)cells overexpressing OAT1 suggested that NCB inhibited the uptake of 6-CFL and lamivudine.Similar results were obtained for NCA and CMC.NCB was a noncompetitive and competitive inhibitor interaction with OAT1.IC50 values of NCB,NCA and CMC for inhibiting OAT1-mediated lamivudine transport were 2.46,8.35 and 0.61μmol·L^–1,respectively.In vivo,the pharmacokinetic results of lamivudine in rats showed that the mean area under the plasma concentration-time curve(AUC0-∞)and maximal plasma concentration(Cmax)of lamivudine after co-administration is increased 2.94-fold and 1.87-fold,respectively,compared to lamivudine administration alone.The results of interactions between lamivudine and these bi-flavone components in Stellera chamaejasme L.extracts via OAT1 in vivo are consistent with studies in vitro.The inhibition of OAT1-mediated uptake of lamivudine by NCB,NCA and CMC is the possible mechanism for Stellera chamaejasme L.extracts improving the oral bioavailability of lamivudine in rats.

pH-triggered cancer-targeting polymers:From extracellular accumulation to intracellular release

Stimuli-responsive polymers are promising to achieve targeted delivery,improved stability during circulation,and controlled release of therapeutic and diagnostic agents.Among them,pH-responsive polymeric nanocarriers have attracted significant attention as pH varies in different body fluids(e.g.,stomach,intestine,and colon)and intracellular organelles(e.g.,endosome,lysosome,and mitochondria)to maintain homeostasis,while distinctive pH changes are also found in certain pathological states.For example,the extracellular environment of the tumor is acidic,which can be employed to drive selective delivery.During the internalization process,since most nanocarriers enter cells upon endocytosis where a drop of pH from 6.5 to 5.0 can occur from endosome to lysosome,pH-sensitive groups have been developed for enhanced cargo release.In this review,both non-covalent and covalent interactions responsive to pH changes are introduced,with a focus on the structure-property relationship and their applications in cancer targeting and endosomal escape.

Emerging new therapeutic antibody derivatives for cancer treatment

Monoclonal antibodies constitute a promising class of targeted anticancer agents that enhance natural immune system functions to suppress cancer cell activity and eliminate cancer cells.The successful application of IgG monoclonal antibodies has inspired the development of various types of therapeutic antibodies,such as antibody fragments,bispecific antibodies,and antibody derivatives(e.g.,antibody-drug conjugates and immunocytokines).