Development of novel NK3 receptor antagonists with reduced environmental impact.

The neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling positively regulates the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective antagonists may suppress the reproductive functions of mammals. For development of novel NK3R antagonists with reduced environmental toxicity, a structure-activity relationship study of an NK3R antagonist, talnetant, was carried out. Among several talnetant derivatives with labile functional groups in the natural environment, 3-mercaptoquinoline 2f exhibited a comparable biological activity to that of the parent talnetant. Additionally, compound 2f was converted into the disulfide 3f or isothiazolone 8 by air-oxidation, both of which showed no binding affinity to NK3R.

Structure-activity relationship study of 4-(thiazol-5-yl)benzoic acid derivatives as potent protein kinase CK2 inhibitors.

Two classes of modified analogs of 4-(thiazol-5-yl)benzoic acid-type CK2 inhibitors were designed. The azabenzene analogs, pyridine- and pyridazine-carboxylic acid derivatives, showed potent protein kinase CK2 inhibitory activities [IC50 (CK2α)=0.014-0.017µM; IC50 (CK2α')=0.0046-0.010µM]. Introduction of a 2-halo- or 2-methoxy-benzyloxy group at the 3-position of the benzoic acid moiety maintained the potent CK2 inhibitory activities [IC50 (CK2α)=0.014-0.016µM; IC50 (CK2α')=0.0088-0.014µM] and led to antiproliferative activities [CC50 (A549)=1.5-3.3µM] three to six times higher than those of the parent compound.

Investigations of possible prodrug structures for 2-(2-mercaptophenyl)tetrahydropyrimidines: reductive conversion from anti-HIV agents with pyrimidobenzothiazine and isothiazolopyrimidine scaffolds.

3,4-Dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182) and 3,4-dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine are the heterocyclic antiretroviral agents against human immunodeficiency virus type 1 (HIV-1) infection. On the basis of similar structure-activity relationships of anti-HIV activities toward the early-stage of viral infection between these unique scaffolds, the transformations under the bioassay conditions were investigated. The distinctive S-N bond in the isothiazolopyrimidine scaffold was immediately cleaved under reductive conditions in the presence of GSH to generate a thiophenol derivative. A similar rapid conversion of PD 404182 into the same thiophenol derivative was observed, suggesting that pyrimidobenzothiazine and isothiazolopyrimidine scaffolds may work as prodrug forms of the common bioactive thiophenol derivatives.

Identification of anti-HIV agents with a novel benzo[4,5]isothiazolo[2,3-a]pyrimidine scaffold.

3,4-Dihydro-2H-benzo[4,5]isothiazolo[2,3-a]pyrimidine is a newly identified antiviral agent against human immunodeficiency virus type 1 (HIV-1) infection, derived from 3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (PD 404182). The introduction of the hydrophobic 8-aryl substituent on the benzene substructure improved its anti-HIV activity, resulting in the identification of 6-fold more potent analogs. In addition, it was demonstrated that these isothiazolopyrimidine derivatives exert anti-HIV effects at an early stage of viral infection.

Structure-activity relationship study of phenylpyrazole derivatives as a novel class of anti-HIV agents.

The structure-activity relationship of phenylpyrazole derivative 1 was investigated for the development of novel anti-HIV agents. Initial efforts revealed that the diazenyl group can be replaced by an aminomethylene group. In addition, we synthesized various derivatives by the reductive amination of benzaldehydes with 5-aminopyrazoles and carried out parallel structural optimization on the benzyl group and the pyrazole ring. This optimization led to a six-fold more potent derivative 32j than the lead compound 1, and this derivative has a 3',4'-dichloro-(1,1'-biphenyl)-3-yl group.

Cellular mRNA expressions of anti-oxidant factors in the blood of preeclamptic women.

OBJECTIVE: To assess the alterations of mRNA expressions associated with oxidative stress in the cellular component of blood from pregnant women with pre-eclampsia. METHODS: Peripheral blood samples were obtained from pregnant women with and without pre-eclampsia. Cellular RNA was subjected to a reverse transcription polymerase chain reaction (PCR) assay in order to examine the mRNA distribution among women with pre-eclampsia (n = 24) and control subjects (n = 24) during 35-41 weeks of gestation. The data were analyzed by non-parametric statistics. RESULTS: Significant differences between the pre-eclampsia subjects and the controls were observed in the gene expressions associated with oxidative stress. Lower values in the pre-eclampsia group were found for heme oxygenase (HO)-1, HO-2, catalase and superoxide dismutase (SOD). The HO-1, HO-2 and the catalase levels significantly correlated with proteinuria, and the HO-2 level with systolic blood pressure. CONCLUSION: Significantly lower concentrations of HO-1, HO-2, SOD and catalase are found in the cellular component of blood from pre-eclamptic patients. The values correlate with the severity of pre-eclampsia. These findings indicate that enhanced oxidative stress and a decrease in the number of anti-oxidant enzymes may be associated with pre-eclampsia.

Prediction of preeclampsia by analysis of cell-free messenger RNA in maternal plasma.

OBJECTIVE: The purpose of this study was to predict the occurrence of preeclampsia in a series of patients at gestational week 15-20 weeks, with the use of a panel of messenger RNA markers. STUDY DESIGN: Data from 62 patients with preeclampsia who were asymptomatic at the time of blood testing and 310 control subjects were analyzed. Multivariable analysis was performed with discriminant analysis. RESULTS: Univariable analysis identified vascular endothelial growth factor receptor 1 as the marker with the highest detection rate; placenta-specific 1 with the lowest. Mean estimated score for preeclampsia was 9.4 for control subjects and 72.5 for subjects who experienced preeclampsia. A receiver operating characteristic curve that was obtained with the estimated score for preeclampsia as a test variable yielded a detection rate of 84% (95% CI, 71.8-91.5) at a 5% false-positive rate with an area under the curve of 0.927 (P < .001). Again, detection rate and score for each patient for classification as preeclamptic correlated with severity. CONCLUSION: A panel of messenger RNA is able to detect subjects who will experience preeclampsia.

Evaluation of physiological alterations of the placenta through analysis of cell-free messenger ribonucleic acid concentrations of angiogenic factors.

OBJECTIVE: Placental messenger ribonucleic acid (mRNA) has been shown to circulate in maternal plasma. We investigated concentrations of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), and endoglin in subjects with preeclampsia, compared with normal pregnancies. STUDY DESIGN: Peripheral blood samples were obtained from preeclampsia (n = 43) and control subjects (n = 41). Plasma ribonucleic acid was subjected to analysis by reverse transcription-polymerase chain reaction assay to examine the mRNA distribution among women with preeclampsia and control subjects during weeks 35-41 of gestation. RESULTS: Concentrations of VEGF, VEGFR-1, and endoglin mRNA of women with preeclampsia were significantly increased. The mRNA values were observed to correlate directly with the severity of hypertension and proteinuria. VEGFR-1 mRNA was markedly elevated in women with preeclampsia and hemolysis, elevated liver enzymes, and low platelet syndrome. CONCLUSION: The mRNA concentrations of VEGF, VEGFR-1, and endoglin were observed to correlate directly with the severity of preeclampsia.

Placenta-derived, cellular messenger RNA expression in the maternal blood of preeclamptic women.

OBJECTIVE: To perform gene expression profiling and real-time quantitative reverse-transcription polymerase chain reaction (PCR) analysis to identify biomarkers of preeclampsia in cellular messenger RNA (mRNA) from maternal blood. METHODS: We performed a microarray analysis with five maternal blood samples from women with preeclampsia and five matched control subjects. Up-regulated gene expression was further analyzed through reverse-transcription PCR analysis with 28 consecutive blood samples from women affected with preeclampsia and 29 controls. RESULTS: Both pregnancy-specific beta1 glycoprotein and trophoblast glycoprotein were selected based on microarray analysis. Reverse-transcription PCR analysis detected significantly increased mRNA concentrations among women in the preeclampsia group. When stratified according to mild or severe preeclampsia, 19.2-fold and 41.8-fold increases in pregnancy-specific beta1 glycoprotein and 8.3-fold and 10.6-fold increases in trophoblast glycoprotein were observed, respectively. Among women with hemolysis, elevated liver enzymes, and low platelet count syndrome, 51.6-fold and 13.1-fold increases in pregnancy-specific beta1 glycoprotein and trophoblast glycoprotein were observed, respectively. In the preeclampsia group, pregnancy-specific beta1 glycoprotein correlated with severity of proteinuria (P<.001) and systolic blood pressure (P=.01). CONCLUSION: The mRNA expression of pregnancy-specific beta1 glycoprotein and trophoblast glycoprotein is up-regulated in cells circulating within blood from women with preeclampsia, and pregnancy-specific beta1 glycoprotein expression is positively correlated with the clinical severity of preeclampsia. LEVEL OF EVIDENCE: II.

Recent advances in non-invasive prenatal DNA diagnosis through analysis of maternal blood.

Prenatal diagnosis of aneuploidy and single-gene disorders is usually performed by collecting fetal samples through amniocentesis or chorionic villus sampling. However, these invasive procedures are associated with some degree of risk to the fetus and/or mother. Therefore, in recent years, considerable effort has been made to develop non-invasive prenatal diagnostic procedures. One potential non-invasive approach involves analysis of cell-free fetal DNA in maternal plasma or serum. Another approach utilizes fetal cells within the maternal circulation as a source of fetal DNA. At the present time, fetal gender and fetal RhD blood type within RhD-negative pregnant women can be reliably determined through analysis of maternal plasma. Furthermore, genetic alterations can be diagnosed in the maternal plasma when the mother does not have the alterations. However, the diagnosis of maternally inherited genetic disease and aneuploidy is limited using this approach. Non-invasive prenatal diagnosis through examination of intact fetal cells circulating within maternal blood can be used to diagnose a full range of genetic disorders. Since only a limited number of fetal cells circulate within maternal blood, procedures to enrich the cells and enable single cell analysis with high sensitivity are required. Recently, separation methods, including a lectin-based method and autoimage analyzing, have been developed, which have improved the sensitivity of genetic analysis. This progress has supported the possibility of non-invasive prenatal diagnosis of genetic disorders. In the present article, we discuss recent advances in the field of non-invasive prenatal diagnosis.

Cell-free mRNA concentrations of CRH, PLAC1, and selectin-P are increased in the plasma of pregnant women with preeclampsia.

OBJECTIVE: To compare mRNA concentrations of corticotrophin-releasing hormone (CRH), placenta specific-1 (PLAC1), and selectin-P in preeclamptic and normal pregnancies. METHODS: Peripheral blood samples were obtained from 43 pregnant women with preeclampsia and 41 control subjects. Plasma was harvested from samples and RNA extracted. Plasma RNA was analyzed using reverse transcription polymerase chain reaction (PCR) assay. Median concentrations of CRH, PLAC1, and selectin-P mRNA in plasma were compared, to assess possible differences in distribution. Data were also stratified and compared according to clinical severity of preeclampsia. Finally, CRH, PLAC1, and selectin-P were plotted against quantitative distributions of blood pressure and proteinuria. RESULTS: All markers were differently distributed between cases and controls. Median values in subgroups correlated with severity of preeclampsia. All markers correlated with both. Selectin-P was identified as the marker with the highest degree of correlation. No correlation was found between any markers in the control group and proteinuria or blood pressure. CONCLUSION: CRH, PLAC1, and selectin-P are distributed differently in preeclampsia cases compared to controls and correlate with signs of preeclampsia.

Cell-free mRNA concentrations of plasminogen activator inhibitor-1 and tissue-type plasminogen activator are increased in the plasma of pregnant women with preeclampsia.

BACKGROUND: Detection of placental mRNA in maternal plasma has been reported in high-risk pregnancies. We attempted to investigate the concentrations of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) mRNA in maternal plasma in preeclampsia. METHODS: Peripheral blood samples were obtained from healthy pregnant women before and after delivery and also from women with or without preeclampsia. Plasma was isolated from these samples, and RNA was extracted. Plasma PAI-1 and tPA mRNA concentrations were then measured by use of reverse transcription PCR assays. The concentrations were converted into multiples of the median (MoM) of the controls adjusted for gestational age. Data were stratified and analyzed according to the clinical severity of preeclampsia and quantitative distribution of blood pressure and proteinuria. RESULTS: The median (minimum-maximum) PAI-1 mRNA MoM values for women with preeclampsia and controls were 2.48 (0.82-8.53) and 1.00 (0.41-2.33), respectively, whereas the median (minimum-maximum) tPA mRNA MoM values were 3.33 (1.01-10.58) and 1.00 (0.95-1.20), respectively. The concentrations of both PAI-1 and tPA mRNA were significantly increased in cases of preeclampsia, compared with controls (P <0.0001). The MoM values of both mRNA species were directly correlated with the severity of preeclampsia and were greatest among a subgroup of hemolysis, increased liver enzymes, and low platelets pregnancies. CONCLUSION: Maternal plasma PAI-1 and tPA mRNAs are significantly increased in patients with preeclampsia and are positively correlated with the severity of preeclampsia.

Clinical potential for noninvasive prenatal diagnosis through detection of fetal cells in maternal blood.

Fetal cells circulate in maternal blood and are considered a suitable means by which to detect fetal genetic and chromosomal abnormalities. This approach has the advantage of being noninvasive. Since the early 1990s, nucleated erythrocytes (NRBCs) have been considered good target cells for a number of techniques, including fluorescence-activated cell sorting and magnetic cell sorting, using antibodies such as anti-transferrin receptor and anti-gamma-hemoglobin antibodies, followed by analysis with fluorescence in situ hybridization or polymerase chain reaction. In the late 1990s, the National Institute of Child Health and Human Development Fetal Cell Isolation Study assessed the reliability of noninvasive prenatal diagnosis of fetal aneuploidy using NRBCs isolated from maternal circulation. This study revealed the limitations of NRBC separation using antibodies specific for NRBC antigens. A more recent study has demonstrated the efficiency and success of recovery of NRBCs using a galactose-specific lectin, based on the observation that erythroid precursor cells have a large quantity of galactose molecules on their cell surface. Thus, recent advances in this field enhance the feasibility of this diagnostic method. This review article focuses on various methods of detection of fetal cells within the maternal circulation, as well as the status of previous and current studies and the prospective view for noninvasive prenatal diagnosis using fetal cells from the maternal circulation.