The PK bioanalysis method study of c-Met antibody-drug conjugate (RC108) in cynomolgus monkey / 药学学报

Antibody-drug conjugate (ADC) has the characteristics of low toxicity and high efficiency, and plays an important role in cancer treatment. However, due to the complexity of its structure, it brings difficulties in pharmacokinetic (PK) bioanalysis. This study established an analytical method for the detection of ADC (RC108) in cynomolgus monkey plasma by ligand-binding assay (LBA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which was used to analyze and quantify the total antibody, bound antibody and free drug in cynomolgus monkey plasma. Based on the LBA method, rabbit anti-RC108 Fab and mouse anti-MMAE (monomethyl auristatin E) mAb were pre-coated in 96-well plates as the total antibody and antibody binding reagents, respectively. The samples to be tested were added, and then the detection reagents were added in turn. Goat anti-human IgG (H+L)-HRP, chromogenic solution tetramethylbenzidine (TMB), H2SO4 terminate the reaction, read data at 450 nm/630 nm wavelength of microplate reader; LC-MS/MS analysis method quantifies MMAE concentration, and refer to relevant regulations for methodological validation. The analytical method for quantifying total antibody, bound antibody and free drug of RC108 drug obtained good accuracy and precision, and the selectivity, dilution linearity, hook effect, parallelism and stability were verified. Meet the requirements of biological analysis. Finally, a bioanalytical method for the determination of the concentration of the test substance RC108 (total antibody, conjugated antibody, free MMAE) in cynomolgus monkey plasma with high sensitivity and high throughput was established by LBA and LC-MS/MS method. Subsequent non-clinical research on PK research in cynomolgus monkeys will provide technical support.

Coagulation modifiers targeting SARS-CoV-2 main protease Mpro for COVID-19 treatment: an in silico approach

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on viral polyprotein processing, catalysed by the main proteinase (Mpro). The solution of the SARS-CoV-2 Mpro structure allowed the investigation of potential inhibitors. This work aims to provide first evidences of the applicability of commercially approved drugs to treat coronavirus disease-19 (COVID-19). We screened 4,334 compounds to found potential inhibitors of SARS-CoV-2 replication using an in silico approach. Our results evidenced the potential use of coagulation modifiers in COVID-19 treatment due to the structural similarity of SARS-CoV-2 Mpro and human coagulation factors thrombin and Factor Xa. Further in vitro and in vivo analysis are needed to corroborate these results.

Expresión génica de ligandos mica, micb y ulbp (1-6) del receptor NKG2D de células natural killer y metaloproteinasas adam10, adam17 y mmp14 en lineas celulares de cáncer de cervical / Gene expression of mica, micb and ulbp (1-6) ligands of the NKG2D receptor of natural killer cells and metalloproteinases adam10, adam17 and mmp14 in cells lines of cervical cancer

RESUMEN El CCU es la segunda causa de muerte en mujeres de nuestro país. Dentro de los primeros mecanismos de defensa del hospedero se encuentra la respuesta inmune de las células NK y su función lítica a expensas de su receptor activador NKG2D, el cual posee como ligandos mica, micb y ulbp (1-6), los cuales se expresan en células transformadas y/o infectadas por virus. Uno de los mecanismos de evasión por parte de la célula tumoral es el clivaje de estas proteínas a través de metaloproteinasas como adam10, adam17 y mmp14. Se analizó la expresión de estos ligandos y metaloproteinasas mediante PCR tiempo real, en lineas celulares de referencia para cáncer cervical como HeLa (positiva para VPH-18) y C33A (negativa para VPH). Se obtuvieron valores representativos de expresion relativa genica con diferencias significativas asi: mmp14 en linea HeLa (p= 0.006); y mica y ulbp-3 en la linea C33A (p= 0.020 y p=0.003 respectivamente). Por lo tanto, se podría sugerir que la expresión de mmp14 se encuentra posiblemente involucrados con la presencia de VPH causante del cancer cervical y la respuesta inmunne innata desarrollada.

ABSTRACT Cervical cancer is the second leading cause of death in women in our country. Within the first host defense mechanisms is the immune response of NK cells and their lytic function at the expense of its NKG2D receptor activator which has as ligands mica, micb and ulbp (1-6), which are expressed in transformed cells and / or virally infected. One of the mechanisms of evasion by the tumor cell is the cleavage of these proteins through metalloproteinases as adam10, adam17 and mmp14. We analyzed the expression of these ligands and metalloproteinases by real time PCR, in reference to cell lines HeLa cervical cancer (positive for HPV-18) and C33A (negative for HPV). We obtained representing relative gene expression with significant differences from the other lines of study as follows: mmp14 in HeLa (p = 0.006); and mica and ulbp-3 in C33A (p = 0.020 and p = 0.003 respectively). Thus one might suggest that the expression of mmp14 is possible involved with HPV presence causing high risk of cervical cancer and innate inmunne response developed.

Characterization of the ligand binding of PGRP-L in half-smooth tongue sole (Cynoglossus semilaevis) by molecular dynamics and free energy calculation

Background: Peptidoglycan (PGN) recognition proteins (PGRPs) are important pattern recognition receptors of the host innate immune system that are involved in the immune defense against bacterial pathogens. PGRPs have been characterized in several fish species. The PGN-binding ability is important for the function of PGRPs. However, the PGRP-PGN interaction mechanism in fish remains unclear. In the present study, the 3-D model of a long PGRP of half-smooth tongue sole (Cynoglossus semilaevis) (csPGRP-L), a marine teleost with great economic value, was constructed through the comparative modeling method, and the key amino acids involved in the interaction with Lys-type PGNs and Dap-type PGNs were analyzed by molecular dynamics and molecular docking methods. Results: csPGRP-L possessed a typical PGRP structure, consisting of five ß-sheets and four α-helices. Molecular docking showed that the van der Waals forces had a slightly larger contribution than Coulombic interaction in the csPGRP-L-PGN complex. Moreover, the binding energies of csPGRP-L-PGNs computed by MM-PBSA method revealed that csPGRP-L might selectively bind both types of MTP-PGNs and MPP-PGNs. In addition, the binding energy of each residue of csPGRP-L was also calculated, revealing that the residues involved in the interaction with Lys-type PGNs were different from that with Dap-type PGNs. Conclusions: The 3-D structure of csPGRP-L possessed typical PGRP structure and might selectively bind both types of MTP- and MPP-PGNs, which provided useful insights to understanding the functions of fish PGRPs.

Molecular and functional analysis of monoclonal antibodies in support of biologics development

Monoclonal antibody (mAb)-based therapeutics are playing an increasingly important role in the treatment or prevention of many important diseases such as cancers, autoimmune disorders, and infectious diseases. Multi-domain mAbs are far more complex than small molecule drugs with intrinsic heterogeneities. The critical quality attributes of a given mAb, including structure, post-translational modifications, and functions at biomolecular and cellular levels, need to be defined and profiled in details during the developmental phases of a biologics. These critical quality attributes, outlined in this review, serve an important database for defining the drug properties during commercial production phase as well as post licensure life cycle management. Specially, the molecular characterization, functional assessment, and effector function analysis of mAbs, are reviewed with respect to the critical parameters and the methods used for obtaining them. The three groups of analytical methods are three essential and integral facets making up the whole analytical package for a mAb-based drug. Such a package is critically important for the licensure and the post-licensure life cycle management of a therapeutic or prophylactic biologics. In addition, the basic principles on the evaluation of biosimilar mAbs were discussed briefly based on the recommendations by the World Health Organization.

Receptor activation of curcumin on human peroxisome proliferators-activated receptors γ1 / 中草药

Objective: To determine whether curcumin is a natural ligand for human peroxisome proliferators-activated receptors γ1 (hPPARγ1) by measuring the combination ability and internal activity. Methods: The combination ability was determined by radioactively labeled ligand binding experiment (RBCA), and the internal activity was estimated by trans-activation reporter gene test. Results: The combination ability of curcumin on hPPARγ1 showed that IC50 was (8.82 ± 0.74) μmol/L, and Ki was 0.72 μmol/L. The internal activity showed that EC50 was 7.3 μmol/L and Emax was 43.3. Conclusion: Curcumin has affinity and intrinsic activity with hPPARγ1, which suggests that curcumin may be a natural ligand of hPPARγ1.

Genetic analysis of a family with complete androgen insensitivity syndrome.

Androgen insensitivity causes impaired embryonic sex differentiation leading to developmental failure of normal male external genitalia in 46 XY genetic men. It results from diminished or absent biological actions of androgens, which is mediated by the androgen receptor (AR) in both the embryo and secondary sexual development. Mutations in the AR located on the X chromosome are responsible for the disease. Almost 70% of affected individuals inherit the mutation from their carrier mother. We hereby report a 10‑year‑old girl with all the characteristics of complete androgen insensitivity syndrome (CAIS). Similar scenario was observed in 3 maternal aunts, Sequencing of the AR gene in all the family members revealed C 2754 to T transition in exon 6. It was concluded that the C 2754 to T transition rendered the AR incapable of both ligand‑binding and activating the transcription and was the cause of CAIS in the patient.

Ligand binding prediction for BRCA1, a key molecule in the pathogensis of breast cancer.

Background : Cell adhesion plays a pivotal role in diverse biological processes that occur in oncogenesis. BRCA1 was the first breast cancer susceptibility gene to be successfully identified and cloned. The role of BRCA1 as a predictive marker of chemotherapy response in breast cancer is proposed. Ligand binding is an important factor in the success of chemoprevention. Materials and Methods : In this work, the author performed a ligand-binding prediction for BRCA1 using a new bioinformatics tool. Results : According to this study, 10 strong possible ligands were identified. Conclusion : These sites can be useful for further drug development studies.

Glutamate receptors as seen by light: spectroscopic studies of structure-function relationships

Ionotropic glutamate receptors are major excitatory receptors in the central nervous system and also have a far reaching influence in other areas of the body. Their modular nature has allowed for the isolation of the ligand-binding domain and for subsequent structural studies using a variety of spectroscopic techniques. This review will discuss the role of specific ligand:protein interactions in mediating activation in the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptors as established by various spectroscopic investigations of the GluR2 and GluR4 subunits of this receptor. Specifically, this review will provide an introduction to the insight gained from X-ray crystallography and nuclear magnetic resonance investigations and then go on to focus on studies utilizing vibrational spectroscopy and fluorescence resonance energy transfer to study the behavior of the isolated ligand-binding domain in solution and discuss the importance of specific ligand:protein interactions in the mechanism of receptor activation.

Investigations of glucocorticoid receptor mutant without ligand binding domain / 医学研究生学报

Objective:To construct expression vector of human glucocorticoid receptor mutant.Methods:cDNA sequence of human glucocorticoid receptor mutant having no LBD was amplificated through RT-nested PCR,PCR product was directionally cloned to pEGFP-N2 plasmid vector,and recombinant plasmids were screened through sequencing.Expressions of recombinant plasmids in human macrophage cell line U937 were observed through fluorescence microscope.Transcriptional activation activity of variant was evaluated through methods of relative activity of luciferase.Results:The 1 601 bp length cDNA was obtained,and result of sequencing confirmed the cloned cDNA sequence is completely coincidence with that of glucocorticoid receptor in Genebank.Expression products of recombinant plasmid mainly located in nucleus.Transcriptional activation activity incresed after recombinant plasmid transfected.Conclusion:Expression vector of glucocorticoid receptor mutant was constructed successfully,which has transcriptional activation activity independent of glucocorticoid.

Density and Affinity of IL-6 Receptors in Human Leukemic Cells / 中国肿瘤生物治疗杂志

Objective: To make a study of density and affinity of IL-6R in human leukemic cell lines, and discuss the affection of high affinity IL-6R to the targeted treatment of leukemia with IL-6-PE40 fusion protein. Methods: Radial binding assay with scatchard plot and FACS were used to analysis the density and affinity of IL-6R and protein expression of IL-6Rα and β subunits in totally 8 representative human leukemic cell lines. Results: Myelocytie, monocytic and erythrocytic leukemic cell lines U937, HL-60, KG1 and TF1 express high affinity IL-6R, whose average density per cell is 2 502,2 874, 2 319 and 9 329 respectively, however no 125I-IL-6 binding was detected on chronic myelocytic leukemic cell line K562 and lymphoblastic leukemic cell lines such as Raji, CEM and HUT28. These results correlate with those of FACS highly. Conclusion:These observations suggest that acute nonlymphoblastic leukemic cells may be more suitable for targeted treatment with IL-6-PFA0 fusion protein.

Estrogen Receptor Analysis on Fine Needle Aspirates and Biopsies from Palpable Breast Carcinoma

The estrogen hormone receptor (ER) content of human breast cancer has assumed an important role as a predictor of hormone therapy response and as a prognostic indicator. The conventional technique is the dextran-coated charcoal (DCC) method or a ligand-binding assay (LBA) based on the measurement of radiolabeled steroids in cytosolic extracts of tissue homogenate. The recent introduction of monoclonal antibodies with high specificity for human ERs has allowed the application of immunocytochemical assays (ICA) in human cancer tissue. An extension of the ICA technique to cytologic specimens is also widely used. Our aim was to evaluate the reliability of ER-ICAs on fine needle aspirates(FNA) from breast cancer patients by comparing it with ER-ICAs and ER-LBAs performed on surgically removed tissues. During a recent 6-month period, ER-ICAs and ER-LBAs were performed in 83 cases. Among these 83 cases, only the 40 cases for which the ER-ICA and the ER-LBA were performed simultaneously ere included in this study. As positive cutoff values, we assumed 10 fmol/mg protein for the ER-LBAs and a semiquantitative score of 4 for the ER-ICAs. The results were as follows : 1) The ER positive rate was 55% (22/40) for ICAs and 47.5% (19/40) for LBAs. The concordance rate between the ER of ICAs and that of LBAs was 82.5% (33/40). 2) The Pearson correlation coefficient between ER-ICAs of fine needle aspirates and that of surgically removed tissue was good (r=0.94, p<0.005) 3) The Spearman correlation coefficient between ER-ICAs of fine needle aspirates and ER-LBAs of surgically removed tissue was good (r=0.57, p=0.0001) In conclusion, ER determination by using the fine needle aspirate is a reliable method in palpable breast cancer. FNA-ER may be a useful method when it is difficult to take sufficient breast cancer tissue, i.e., in cases of diffusely recurrent cancer, liver metastasis, malignant pleural effusion, etc.

The study about the mechanism of antidepressant effect of 7903 / 中国药理学通报

The influences of the N -Isosuccinyl - dichloro - cinnamoylamide (7903) on the NA and 5 -HT systems in the brain were studied. Acute administration of 7903 could significantly enhance the level of NA and 5 -HT in mice brain and cortex of rats, and the level of 5 -HT in the hyporthalamus of rats was tending to be enhanced. However, chronic administration of 7903 (two weeks ) could decrease the level of NA and the ra-tio of 5-HIAA/5-HT in the cortex of rats; Chronic administration of 7903 could also decrease the turnover of NA and 5- HT in mice brain, but had no effects on the special binding of ? - receptor to 3-H - DHA and that of 5 -HT receptor to H - ketanserine in the cortex of rats.

Fluorescence studies on the interaction of some ligands with carcinoscorpin, the sialic acid specific lectin, from the horseshoe crab, Carcinoscorpius rotundacauda.

The binding affinities of some ligands towards the sialic acid-specific lectin carcinoscorpin, from hemolymph of the horseshoe crab Carcinoscorpius rotundacauda have been determined by protein fluorescence quenching in presence of ligands. Among the ligands studied, the disaccharide O-(N-acetylneuraminyl)-(2→6)-2-acetamido-2-deoxy-Dgalactitol has the highest Ka(l.15 × 106 M–1) for carcinoscorpin. Studies on the effect of pH on Ka values of disaccharide suggests the possible involvement of amino acid residues having pKa values around 6.0 and 9.0 in the binding activity of carcinoscorpin. There were distinct changes in the accessibility of the fluorescent tryptophan residues of carcinoscorpin by ligand-binding as checked through potassium iodide quenching.