Haifeng Song

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study.

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro

AbstractAim:To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion.Methods:Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-127I iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC50) of sifuvirtide on cell fusion was determined by co-cultivation assay.Results:The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88–5000 μg/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626±2 886 μg/L and 528±191 μg/L, and the terminal elimination half-lives (T1/2) were 6.3±0.9 h and 5.5±1.0 h, respectively. After sc, Tmax was 0.25–2 h, and the absolute bioavailability was 49%±13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC50 of 0.33 μg/L.Conclusion:An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T1/2 of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC50 in vitro.

Gankyrin is essential for hypoxia enhanced metastatic potential in breast cancer cells

Hypoxia, a critical regulator of tumor growth and metastasis, induces the transcriptional activation of several pathways involved in proliferation, migration and invasion. Gankyrin was found to be overexpressed, and also promoted the metastasis in breast cancer cells, which is also involved in the regulation of hypoxia inducible factor‑1 and hypoxia‑inducible factor‑1α. The present study showed that gankyrin mRNA and protein expression were increased under hypoxic conditions in the BT474 breast cancer cell line, accompanied with increased ability of cell migration and invasion. Lentivirus‑mediated siRNA targeting gankyrin was transfected into BT474 cells. Wound‑healing and transwell experiments showed that gankyrin deletion abrogated the increased migration and invasion of BT474 cells due to hypoxia. In addition, E‑cadherin was found to be involved in the gankyrin induced invasion of breast cancer cells due to hypoxia. The present study indicated that gankyrin deletion abrogated the increased metastatic potential of breast cancer cells under hypoxic conditions partly through regulating E‑cadherin, suggesting that an improved understanding of gankyrin may offer a potential therapeutic target for the treatment of human breast cancer metastasis.

An improved on-line solid phase extraction coupled HPLC-MS/MS system for quantification of sifuvirtide in human plasma.

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1-6250 ng/mL, and the correlation coefficients (r(2)) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from -7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC-MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).

Structure-efficacy relationships of immunostimulatory activity of CpG-containing oligodeoxynucleotides on mouse spleen cells.

AbstractAim:To study the relationship between primary structures of oligodeoxynucleo-tides (ODN) containing unmethylated deoxycytidyl-deoxyguanosine (CpG) dinucleotide motifs and their immunostimulatory activities in mouse spleen cells.Methods:A series of CpG ODN with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cell proliferation were determined by [3H]thymidine incorporation assay. Cytokine (interleukin [IL]-6, IL-12, and IFN-α) secretion spectra induced by CpG ODN were assessed by ELISA. The ability of CpG ODN to activate natural killer cells was evaluated by standard 4 h 51Cr-release assay. Flow cytometry was utilized to examine the expressions of various lymphocyte surface molecules on diverse immunocytes. An effective CpG ODN for murine, ODN1826, was set as the template of modification and the positive control.Results:The immunostimulatory activities of CpG ODN with different sequences and compositions varied markedly, both in character and in extent. It was useless for improving the immunostimulatory activity of ODN1826 by simply increasing the functional hexameric CpG motif number, modifying the site of CpG motifs, or changing the distance between multi-CpG motifs. However, an addition of a self-complementary palindrome structure at the 3′-end, but not the 5′-end of CpG ODN, aroused marked improvement in its activity. Several designed ODN had superior comprehensive immunostimulatory properties compared to ODN1826.Conclusion:The immunostimulatory activity of a CpG ODN was relevant to its primary structure. It was useless for promoting immunostimulatory activity to simply change CpG motif number, space, or distance. The 3′-end palindrome structure of CpG ODN is associated with enhanced immunostimulatory activity.

Optimal design and validation of antiviral siRNA for targeting hepatitis B virus

AbstractAim:Optimal design of antiviral short-interfering RNA (siRNA) targeting highly divergent hepatitis B virus (HBV) was validated by quantitative structure-activity relationship (QSAR) analysis.Methods:The potency of 23 synthetic siRNAs targeting 23 sites throughout HBV pregenomic RNA were evaluated at 10 nmol/L by determining the inhibition on the expression of S/P/pregenomic mRNA and hepatitis B surface antigen (HBsAg) quantitatively in HepG2.2.15 cells. Genotype homology within HBV genomes was identified through plentiful computational analysis and the multiple linear regression analysis was made to validate the relationship between the functional siRNAs and primary characteristics. Based on the preliminary results, relationships between different determined endpoints [S/P mRNA, HBsAg, C/P mRNA, hepatitis B e antigen (HBeAg) and viral DNA load] and siRNA efficacy evaluation were investigated.Results:Genotype homology, open reading frame (ORF) S/P, X and C had tight correlation with the ability of siRNAs on inhibiting the expression of S/P/Pregenomic mRNA and HBsAg (P<0.01), of which, ORF C was negatively correlated with the siRNA potency (P<0.05). Further study showed that siRNA potency evaluation was influenced by different determined endpoints. P-target siRNAs showed significant inhibition on the S mRNA and HBsAg expression. S-target siRNAs inhibited the expression of S mRNA and HBsAg strongly. X-target siRNAs played active roles in inhibiting all 5 determined endpoints. C-target siRNAs blocked the expression of C mRNA, HBeAg and viral DNA load significantly.Conclusion:The antiviral potency of siRNA was relevant to its primary characteristics and determined endpoints were important for siRNA efficacy evaluation for complex genome with overlapping ORF, which was helpful for siRNA optimal design.

Immunostimulatory and anti-neoplasm effects of a novel palindrome CpG oligodeoxynucleotide in mice

Aim:DNAs containing unmethylated CpG motifs can stimulate innate and adaptive immunity. The aim of this study was to investigate the immunostimulatory and anti-neoplasm effects of a novel CpG oligodeoxynucleotide, ODN10, in tumor-bearing mice.Methods:B16 melanoma-bearing C57BL/6 mice were administered ip or sc with ODN10 or conventional CpG ODN1826 on the indicated days post inoculation. The animal survival rate and the inhibitory effect on tumor growth were observed in vivo. B and T lymphocyte proliferation, natural killing cell cytotoxicity and the phagocytic ability of peritoneal macrophages from the animals were determined using [3H]-thymidine incorporation assay, 4-h 51Cr release assay and neutral red chromometry method, respectively. The serum levels of IL-12, IL-4 and IgE were quantified using ELISA assays. Histological examination of tumor tissues was performed after HE staining, and the expression of PCNA, CD63, and CD80 in tumor tissues was analyzed with immunohistochemistry.Results:ODN10 (1, 5 and 25 mg/kg) significantly inhibited the growth and metastasis of the tumor, and significantly prolonged the survival of tumor-bearing mice, as compared with ODN1826. The immune status was suppressed in tumor-bearing mice. Both ODN10 and ODN1826 significantly reversed the suppressed immunoactivities in tumor-bearing mice, which included promoting B and T lymphocyte proliferation, enhancing NK cell and peritoneal macrophage activities, inducing IL-12 secretion and inhibiting IL-4 and IgE secretion. Further, CpG ODNs decreased PCNA and CD63 expression while induced expression of CD80. ODN10 presented more potent activity, and displayed the most prominent immunostimulatory potential.Conclusion:ODN10 produces prominent immunomodulatory effects on cellular immunity in tumor-bearing mice, which might help reverse the established Th2-type responses to the Th1-type responses, thus may be used as a potent anti-tumor immunotherapy agent or adjuvant.

Preclinical evaluation of the efficacy, pharmacokinetics and immunogenicity of JS-001, a programmed cell death protein-1 (PD-1) monoclonal antibody

JS-001 is the first monoclonal antibody (mAb) against programmed cell death protein-1 (PD-1) approved by the China Food and Drug Administration (CFDA) into the clinical trails. To date, however, no pre-clinical pharmacological and pharmacokinetic (PK) data are available. In this study, we investigated the efficacy of JS-001 and conducted a preclinical PK study, including the monitoring of anti-drug antibodies (ADAs). We found that JS-001 specifically bound to PD-1 antigen with an EC50 of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC50 of 3.0 and 3.1 nmol/L, respectively. Furthermore, JS-001 displayed distinct species cross-reactivity: it could bind to the PD-1 antigen on the peripheral blood mononuclear cells (PBMCs) of humans and cynomolgus monkeys, but not to those of mice and woodchucks; the Kd values for the interaction between JS-001 and PD-1 antigens on CD8+ T cells of human and cynomolgus monkey were 2.1 nmol/L and 1.2 nmol/L, respectively. In vitro, treatment with JS-001 (0.01–10 μg/mL) dose-dependently stimulated human T cell proliferation, as well as IFN-γ and TNF-α secretion. In HBsAg-vaccinated cynomolgus monkeys, the expression of PD-1+/CD4+ and PD-1+/CD8+ was significantly elevated, intramuscular injection of JS-001 (1 and 10 mg/kg) resulted in dramatic decreases in PD-1+/CD4+ and PD-1+/CD8+ expression in a dose-dependent manner, which was supported by PD-1 receptor occupancy (RO) results. In the PK study, 18 cynomolgus monkeys treated with single, ascending doses of 1, 10, and 75 mg/kg, and another 6 cynomolgus monkeys received 10 mg/kg successive administration. The plasma clearance of JS-001 followed a linear PK profile with single administration in the 1 and 10 mg/kg groups and a non-linear PK profile in the 75 mg/kg group. In the successive 10 mg/kg administration group, no drug accumulation was observed. But the AUC from the last exposure was lower than that of the first administration, which was probably due to the production of ADAs, as demonstrated in immunogenicity study. These non-clinical data are encouraging and provide a basis for the efficacy and safety of JS-001 in clinical trials.

Enhancing efficacy and mucosa-tropic distribution of an oral HIV-PsV DNA vaccine in animal models.

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