Effect of genetic polymorphisms on the pharmacokinetics of gefitinib in healthy Chinese volunteers.

Gefitinib is the first-generation drug of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) metabolised by the cytochrome P450 and transported by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). In the present study, the pharmacokinetics of gefitinib in healthy Chinese volunteers was investigated and the effect of genetic polymorphisms on its variability was evaluted.Forty-five healthy volunteers were administered a single dose of gefitinib and the blood samples were used for quantifying the concentration of gefitinib and genotyping fifteen single-nucleotide polymorphisms of cytochrome P450 enzymes (CYP3A4, CYP3A5, CYP2D6, CYP2C9 and CYP2C19) and drug transporters (ABCB1 and ABCG2).CYP3A5*3 (rs776746) polymorphism showed a significant influence, with higher gefitinib AUC0-t in carrier of CC genotype than in CT/TT genotype (BH-adjusted p value <0.05). For CYP2C9*3 (rs1057910), significant differences in pharmacokinetics of gefitinib were detected between carriers of AA and AC genotypes, with higher AUC0-t, AUC0-∞ and Cmax in carrier of AC genotype than in AA gen-otype (BH-adjusted p value <0.05). No associations were found between SNPs in CYP3A4, CYP2D6, CYP2C19, ABCB1, ABCG2 and the pharmacokinetics of gefitinib.The SNPs in CYP3A5*3 (rs776746) and CYP2C9*3 (rs1057910) were found to be associated with altered gefitinib pharmacokinetics in healthy Chinese volunteers.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts.

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

Multiorgan engraftment and differentiation of human cord blood CD34+ Lin- cells in goats assessed by gene expression profiling.

To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+ Lin- cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45-55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2-36% of all cells examined). We identified human beta2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions.

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.

[Analysis of human cells in transplanted goats using fluorescence in situ hybridization].

OBJECTIVE: To analyze the existence and the dynamic cell frequencies of human cells in goats transplanted in utero with human hematopoietic stem cell (hHSC) by using fluorescence in situ hybridization (FISH) technique. METHODS: Interphase FISH (IFISH) with human-specific 17-chromosome satellite DNA and/or human-specific Y-chromosome satellite DNA as probes was performed to analyze the presence and proportions of human cells in 13 transplanted goats. Samples were peripheral blood cells, bone marrow smears and liver touch imprint preparations. RESULTS: Of the 13 transplanted goats, eleven were identified to present human cells. Among them, two goats transplanted with human male HSC were found to have human male cells. The results demonstrated that these transplanted goats were human/goat HSC xenogeneic chimeras. Human cell frequencies decreased with the goat age (months), but the longest survival reached 21 months. During the detected life periods of goats, human cell frequencies in peripheral blood, bone marrow and liver tissues were less than 1@1000, but local human cell frequencies of 207.92@1000 and 392.41@1000 were detected in the liver tissues of 2 transplanted goats. CONCLUSIONS: The existence and long-term survival of human cells in transplanted goats detected by FISH indicated that goats were appropriate recipients for hHSC in utero transplantation. The lower human cell frequencies in blood and bone marrow, and the higher local human cell frequencies in liver tissues suggested that the microenvironment of goat liver tissues might favor the survival, proliferation and differentiation of human cells.