Publications by authors named "Li-hou Dong"

9 Publications

  • Page 1 of 1

Development and Validation of a Simoa Assay for Determination of Recombinant Batroxobin in Human Serum.

Curr Med Sci 2021 Jun 25;41(3):618-625. Epub 2021 Jun 25.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center of Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, 102206, China.

Recombinant batroxobin (S3101) is a thrombin-like serine protease that binds to fibrinogen or is taken up by the reticuloendothelial system. A literature survey showed no adequate method that could determine sufficient concentrations to evaluate pharmacokinetic parameters for phase I clinical studies. Therefore, a sensitive method is urgently needed to support the clinical pharmacokinetic evaluation of S3101. In this study, a sensitive bioanalytical method was developed and validated, using a Quanterix single molecular array (Simoa) assay. Moreover, to thoroughly assess the platform, enzyme-linked immunosorbent assay and electrochemiluminescence assay were also developed, and their performance was compared with that of this novel technology platform. The assay was validated in compliance with the current guidelines. Measurements with the Simoa assay were precise and accurate, presenting a valid assay range from 6.55 to 4000 pg/mL. The intra- and inter-run accuracy and precision were within -19.3% to 15.3% and 5.5% to 17.0%, respectively. S3101 was stable in human serum for 280 days at -20°C and -70°C, for 2 h prior to pre-treatment and 24 h post pre-treatment at room temperature (22°C-28°C), respectively, and after five and two freeze-thaw cycles at -70°C and -20°C, respectively. The Simoa assay also demonstrated sufficient dilution linearity, assay sensitivity, and parallelism for quantifying S3101 in human serum. The Simoa assay is a sensitive and adequate method for evaluating the pharmacokinetic parameters of S3101 in human serum.
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http://dx.doi.org/10.1007/s11596-021-2382-6DOI Listing
June 2021

Method development and validation of LC-MS/MS-based assay for the simultaneous quantitation of trastuzumab and pertuzumab in cynomolgus monkey serum and its application in pharmacokinetic study.

Biomed Chromatogr 2020 Dec 28;34(12):e4903. Epub 2020 Sep 28.

State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China.

We present a simple and robust LC-MS/MS assay for the simultaneous quantitation of an antibody cocktail of trastuzumab and pertuzumab in monkey serum. The LC-MS/MS method saved costs, decreased the analysis time, and reduced quantitative times relative to the traditional ligand-binding assays. The serum samples were digested with trypsin at 50°C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated using a C18 column (2.1 × 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC-MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 μg/mL. The intra- and inter-assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single-dose intravenous drip administration to cynomolgus monkeys.
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http://dx.doi.org/10.1002/bmc.4903DOI Listing
December 2020

Receptor occupancy measurement of anti-PD-1 antibody drugs in support of clinical trials.

Bioanalysis 2019 Jul 8;11(14):1347-1358. Epub 2019 Aug 8.

State Key Laboratory of Proteomics, Beijing Institute of Lifeomics, Beijing 102206, PR China.

The reliable measurement of receptor occupancy (RO) provides informative data for efficacy and safety evaluation. This study aimed to assess factors affecting RO measurement of anti-PD-1 antibodies in clinical studies. RO performance was assessed using different T-cell activation markers measured by flow cytometry. The validated methodology was then used in support of a clinical study. The optimized active cell population was comprised of CD45RO or CD45RA T cells. The bioanalytical method was validated for inter- and intra-assay precision (coefficient of variation ≤30%) and sample storage stability for 3 days. Consistent RO saturation was observed in Phase Ia clinical trial, although receptor regulation appeared to be different. The formation of anti-drug antibodies had markedly influenced pharmacokinetics and RO. RO measurement in combination with pharmacokinetics and anti-drug antibodies data could allow the integrated evaluation and better understanding of efficacy and safety.
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http://dx.doi.org/10.4155/bio-2019-0090DOI Listing
July 2019

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

Acta Pharmacol Sin 2017 May 20;38(5):710-718. Epub 2017 Mar 20.

Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.

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 EC of 21 nmol/L, and competently blocked the binding of PD-1 antigen to PD-L1 and PD-L2 with IC 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 K 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.
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http://dx.doi.org/10.1038/aps.2016.161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457696PMC
May 2017

BMP-7 attenuates TGF-β1-induced fibroblast-like differentiation of rat dermal papilla cells.

Wound Repair Regen 2013 Mar-Apr;21(2):275-81. Epub 2013 Feb 25.

Department of Burns, First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.

Dermal papilla cells (DPCs) show phenotypic plasticity during wound healing. The multipotency of DPCs is well recognized, but the signaling pathways that regulate the differentiation of these cells into fibroblasts are poorly understood. A preliminary experiment showed that transforming growth factor beta1 (TGF-β1) can induce DPCs to differentiate into fibroblast-like cells, which suggests that DPCs may be a source of wound-healing fibroblasts. Bone morphogenetic protein-7 (BMP-7), a member of the TGF-β superfamily, can prevent and reverse fibrosis by counteracting the TGF-β1-mediated profibrotic effect. To determine whether BMP-7 attenuates the TGF-β1-induced differentiation of DPCs into fibroblasts, we established an in vitro system for DPC differentiation and recorded the gene expression patterns that distinguished DPCs from fibroblasts. The proportion of fibroblast-like cells was significantly enhanced in DPCs treated with TGF-β1, as evidenced by immunocytochemistry, flow cytometry, quantitative real-time reverse transcriptase polymerase chain reaction, and Western blot analysis. BMP-7 and TGF-β1 administration substantially decreased fibroblast-like differentiation, indicating inhibition of TGF-β1-induced differentiation. The antagonistic BMP-7- and TGF-β1-activated signaling pathways can be used to promote wound healing or suppress hypertrophic scarring.
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http://dx.doi.org/10.1111/wrr.12015DOI Listing
October 2013

Differentiation of rat dermal papilla cells into fibroblast-like cells induced by transforming growth factor β1.

J Cutan Med Surg 2012 Nov-Dec;16(6):400-6

Department of Burns,Yichang Hospital of Traditional Chinese Medicine, Clinical College of Traditional Chinese Medicine of China, Three Gorges Univerity, Yichang.

Background: The origin of wound-healing fibroblasts is still debated. Dermal papilla cells (DPCs), which are an important population of stem cells for the regeneration of hair follicles, play a considerable role in cutaneous wound healing. Based on the plasticity of DPCs in wound healing, we hypothesized that DPCs may contribute to the fibroblast population of wound repair.

Objective: To explore the possibility of differentiation of DPCs into fibroblasts induced by transforming growth factor β1 (TGF-β1).

Methods: The fourth passage DPCs were treated with TGF-β1 (10 ng/mL) for 4 days, and a series of methods was used to observe morphologic changes under an inverted phase contrast microscope, to validate the messenger ribonucleic acid expression change in α-smooth muscle actin (α-SMA) and vimentin by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR), to analyze the expression of α-SMA and vimentin protein by flow cytometry, and to semiquantitatively measure the expression of fibroblast-specific protein 1 (FSP1) by Western blot.

Results: DPCs treated with TGF-β1 presented fibroblast-like changes in morphology and immunocytochemistry. The effects of TGF-β1 on α-SMA and vimentin in DPCs were detected on both the transcriptional and the posttranscriptional levels. The results showed that TGF-β1 significantly downregulated α-SMA expression and enhanced the expression of vimentin at all times tested. Further study revealed that TGF-β1 could gradually promote the expression of FSP1 in a time-dependent manner.

Conclusion: DPCs experienced the changes in molecular marker expression in response to TGF-β1, which may be a key source of fibroblasts in wound healing.
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http://dx.doi.org/10.1177/120347541201600608DOI Listing
April 2013

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

Acta Pharmacol Sin 2012 Aug 25;33(8):1047-54. Epub 2012 Jun 25.

Department of Pharmacology and Toxicology, Beijing Institute of Radiation Medicine, Beijing 100850, China.

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 [(3)H]-thymidine incorporation assay, 4-h (51)Cr 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.
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http://dx.doi.org/10.1038/aps.2012.54DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011330PMC
August 2012

Attenuation of alpha1 collagen production with antisense ribonucleic acid in cultured hypertrophic scar fibroblasts.

J Cutan Med Surg 2009 May-Jun;13(3):129-33

Background: It has been demonstrated that hypertrophic scar fibroblasts (HSFs) overexpress collagen messenger ribonucleic acid (mRNA) and protein, especially alpha1 collagen. Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression, suggesting that antisense ribonucleic acid (RNA) can effectively downregulate the expression of alpha1 collagen gene and attenuate the scars.

Aims: This study was conducted to observe the effect of recombinant plasmid pREP9-COL1 on alpha1 collagen expression in HSFs and clarify the prospect of antisense RNA on scar treatment.

Methods: The alpha1 collagen gene fragment including the region of 5' UTR to exon (229 bp) was cloned in the eukaryotic expression plasmid pREP9 in the antisense orientation relative to the RSV-LTR promoter to reconstruct the pREP9- COL1 plasmid. Then it was transferred into HSFs through lipofectamine. The expression of alpha1 collagen was examined by immunostaining, reverse-transcriptase polymerase chain reaction, and Western blots.

Results: The recombinant plasmid pREP9-COL1 with a correct sequence was constructed successfully; pREP9-COL1 consistently inhibited human alpha1 collagen gene expression at both mRNA and protein levels.

Conclusions: Antisense RNA was effective in downregulating alpha1 collagen expression of HSFs. Therefore, this approach offered a prospect of scar treatment by attenuation of alpha1 collagen production with antisense RNA.
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http://dx.doi.org/10.2310/7750.2008.07094DOI Listing
September 2009

Basic fibroblast growth factor (bFGF) alleviates the scar of the rabbit ear model in wound healing.

Wound Repair Regen 2008 Jul-Aug;16(4):576-81

Department of Burns, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.

Studies suggest a possible antiscarring effect of basic fibroblast growth factor (bFGF) during wound healing. However, little is known about the precise pathological mechanisms of bFGF. In particular, there is only limited information available about the mechanism of exogenous administration of bFGF to scar formation. To investigate the effect of bFGF on the hypertrophic scar in the rabbit ear model and to clarify the mechanisms of bFGF on treatment for scar in wound healing, the rabbit ear model of wound healing was created and treated topically with bFGF once daily for 3 months; then we examined the changes of macroscopic and histopathological characteristics of scars and the expression of collagen and collagenase-1 (matrix metalloproteinase-1). The results of macroscopic and histologic characteristics revealed a significant difference between scars treated with bFGF and control scars. The expression of collagen in the scars treated with bFGF was decreased, as compared with the scars treated with saline. Further study revealed that bFGF could remarkably enhance expression of matrix metalloproteinase-1. bFGF could improve the quality of wound healing and remarkably alleviate the scar in the rabbit ear model in wound healing, which suggests that bFGF exerted a net negative effecton scar formation in wound healing. The evidence should contribute to a better understanding of the biological activities of bFGF during hypertrophic scar formation.
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http://dx.doi.org/10.1111/j.1524-475X.2008.00405.xDOI Listing
September 2008
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