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The interactions between tumor immune microenvironment (TIME) and pancreatic cancer cells can affect chemotherapeutic efficacy; however, the mechanisms still remain largely unknown. Thirty items in TIME were comprehensively screened by using tissue microarray from pancreatic cancer patients. Their expressions, interconnections and predictive roles for survival were analyzed. Twenty-one of 30 items could stratify the survival of the patients; however, multivariate analysis found that only 5 independent risk factors could predict worse survival (M2-polarized tumor-associated macrophages (TAMs), IgG4 positive cells, TGF-β1, GM-CSF and lymphangiogenesis). They had a much higher expression levels in tumoral tissue, compared to peritumoral tissue. The Spearman analysis showed that M2-polarized TAM, TGF-β1 and GM-CSF were positively correlated with pancreatic cancer stem cells (PCSC), angiogenesis and lymphangiogenesis. Both human and murine pancreatic cancer cells could induce M2-polarized TAM, which showed substantial roles to decease chemotherapeutic effects. After treated by gemcitabine, both human and murine pancreatic cancer cell lines expressed higher level of immune check points, PCSC markers and varieties of immunosuppressive factors; however, TGF-β1 and GM-CSF had the highest increase. Based on the above results, TGF-β1 and GM-CSF were proposed to be the optimal potential targets to improve chemotherapeutic effects. In immunocompetent murine models, we demonstrated that combined blockade of TGF-β1 and GM-CSF improved the chemotherapeutic effects by inhibition of M2-polarized TAM and induction of CD8 positive T cells. This study presents a novel promising combined strategy to improve the chemotherapeutic effects for pancreatic cancer.

Purpose: Icotinib hydrochloride {4-[(3-ethynylphenyl)amino]-6,7-benzo-12-crown-4-quinazoline hydrochloride}, a novel epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), was designed for the treatment of non-small cell lung cancer (NSCLC). In the present study, we investigated the influence of the CYP2C19*2 and CYP2C19*3 alleles on the pharmacokinetics of icotinib in healthy Chinese volunteers., Methods: In a single-dose pharmacokinetic study, 12 healthy Chinese volunteers received an oral dose of 600 mg of icotinib. Plasma was sampled for up to 72 h post-dose, followed by quantification of icotinib by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS-MS)., Results: Five subjects genotyped as homozygous extensive metabolizers (CYP2C19*1/*1), 6 subjects genotyped as heterozygous extensive metabolizers (CYP2C19*1/*2 or CYP2C19*1/*3), and 1 subject genotyped as a poor metabolizer (CYP2C19*2/*3) and was withdrawn from the research because of urticaria. The mean icotinib AUC(0-∞) and C(max) (14.56 ±5.31 h mg/L and 2.32 ± 0.49 μg/mL) in homozygous EMs was 1.56 and 1.41-fold lower than that in heterozygous EMs (22.7 ± 6.11 and 3.28 ± 0.48, P = 0.046 and 0.047). The mean CL/F (44.18 ± 12.17 L/h) in homozygous EMs was 1.55-fold higher than that in heterozygous EMs (28.42 ± 9.23 L/h, P = 0.013)., Conclusions: The data showed that the pharmacokinetics of icotinib differ significantly between homozygous EMs and heterozygous EMs in CYP2C19.

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202-->109, 232-->93, 282-->202 and 378-->202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between -11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

We developed a rapid, specific and sensitive method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) to determine icotinib concentrations in human plasma and urine. Liquid-liquid extraction (LLE) and direct dilution were firstly used to isolate icotinib from plasma and urine followed by injection of the extracts onto a C(18) column with gradient elution. Ionization of icotinib and midazolam (internal standard, IS) was performed using an electrospray ionization source in positive mode and detection was carried out in multi-reaction monitoring (MRM) mode. The lower limits of quantitation (LLoQ) of icotinib in human plasma and urine by this method were 0.1 and 1.00ng/mL, respectively. The accuracy, precision, specificity, recovery, matrix effect, linearity and several of stabilities have been validated for icotinib in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirement of clinical pharmacokinetic study of icotinib hydrochloride in Chinese healthy subjects.