Preclinical characterization of a non-peptidomimetic HIV protease inhibitor with improved metabolic stability.

Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.

Design, synthesis and characterizations of prodrugs of brexanolone.

A series of prodrugs of brexanolone, the synthetic version of the endogenously produced γ-aminobutyric acid A receptors positive allosteric modulator allopregnanolone, were designed, synthesized, and evaluated in vitro and in vivo. The effect of different function groups connecting to brexanolone C3 hydroxyl as well as those at the chain terminals of prodrug moieties were explored. Through these efforts, prodrugs that can efficiently release brexanolone in vitro and in vivo, and possess a potential for sustained delivery of a long acting brexanolone were discovered.

[Hepcidin and Erythroferrone Levels in Child-Bearing Women with Iron Deficiency Anemia].

OBJECTIVE: To detect serum hepcidin and erythroferrone levels in child-bearing women with iron deficiency anemia (IDA), and to investigate the association between them and iron status parameters. METHODS: The study consisted of 65 child-bearing women (35 with iron deficiency anemia and 30 age-matched healthy women). The levels of serum iron were detected by using automated chemistry analyzer, the contents of serum ferritin were detected by electrochemiluminescence immunoassay, and the levels of serum erythroferrone and hepcidin were detected by specific enzyme-linked immunosorbent assay (ELISA) kit. The quantitative variables between two groups were compared and analyzed by SPSS22.0 software. Spearman correlation was used to detect correlation between the parameters. RESULTS: The levels of Hb, serum iron, ferritin and transferrin saturation were significantly decreased in IDA patients as compared with in control group (P<0.001). Serum hepcidin levels in IDA patients were significant lower than those in control group (P<0.001). Serum erythroferrone levels slightly increased in IDA group (P>0.05). In IDA patients, serum hepcidin concentrations were positively correlated with hemoglobin concentration, serum iron, serum ferritin and transferrin saturation (r=0.448, r=0.496, r=0.754, r=0.491). But, serum erythroferrone concentrations showed no correlation with hemoglobin concentration, serum iron, serum ferritin, transferrin saturation and hepcidin (P>0.05). CONCLUSION: Serum hepcidin levels were significantly decreased in child-bearing women with IDA, but the serum erythroferrone levels were not obviously different between two groups, suggesting that serum erythroferrone may be not involved in the regulation of iron metabolism in child-bearing women with mild and moderate IDA.

Wogonin inhibits in vitro herpes simplex virus type 1 and 2 infection by modulating cellular NF-κB and MAPK pathways.

BACKGROUND: Wogonin, a natural flavonoid-like chemical compound, exhibits anti-inflammatory, antitumor, antiviral, neuroprotective, and anxiolytic effects by modulating a variety of cellular signaling pathways including PI3K-Akt, p53, nuclear factor κB (NF-κB), mitogen-activated protein kinase (MAPK) pathways. In this study, its antiviral effect against herpes simplex virus (HSV) type 1 and 2 (HSV-1 and HSV-2) replication was investigated. RESULTS: Wogonin suppressed HSV-2-induced cytopathic effect (CPE) and reduced viral mRNA transcription, viral protein synthesis, and infectious virion particle titers in a dose-dependent manner. A time-of-drug-addition assay demonstrated that wogonin acted as a postentry viral inhibitor. Wogonin also significantly reduced HSV-induced NF-κB and MAPK pathway activation, which has previously been demonstrated to be important for viral replication. CONCLUSIONS: Our results suggest that the anti-herpes effect of wogonin may be mediated by modulation of cellular NF-κB and JNK/p38 MAPK pathways and imply that wogonin may be useful as an anti-HSV agent.

Complement C3 gene polymorphisms are associated with lipid levels, but not the risk of coronary artery disease: a case-control study.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide. Previous studies have shown that complement component 3 (C3) is associated with atherosclerosis and cardiovascular risk factors. METHODS: We conducted this study to evaluate the associations between tagSNPs in the C3 gene locus and the CAD susceptibility and lipid levels in the Chinese population. A hospital-based case-control study, including 1017 subjects (580 CAD patients and 437 non-CAD controls), was conducted. TagSNPs in the C3 gene were searched and genotyped by using the polymerase chain reaction-ligase detection reaction method. RESULTS: The C3 levels were positively associated with the low-density lipoprotein cholesterol (LDL-C) levels (r = 0.269, P = 0.001). Compared with those in controls, the serum C3 levels in CAD patients were significantly higher (Control: 0.94 + 0.14 g/l; CAD: 1.10 + 0.19 g/l, P < 0.001). No significant differences in genotype or allele frequencies were observed between CAD patients and controls. The minor T allele of rs2287848 was associated with low apolipoprotein A1 (ApoA1) levels in controls (Bonferroni corrected P, Pc = 0.032). Linkage disequilibrium and haplotype analysis established two haplotype blocks (Block1: rs344555-rs2277984, Block 2: rs2287848-rs11672613) and six haplotypes. No significant associations between haplotypes and the risk of CAD were observed (all Pc > 0.05). CONCLUSIONS: The results revealed that C3 gene polymorphisms were associated with the lipid levels, but not CAD susceptibility in the Chinese population.

Relationship between warfarin dosage and international normalized ratio: a dose-response analysis and evaluation based on multicenter data.

PURPOSE: The objectives of the study were to establish a dose-response model for warfarin based on the relationship between daily warfarin dose and international normalized ratio (INR) and to evaluate the stability and reliability of the established model using external data. METHODS: Clinical data were recorded from 676 outpatients with a steady-state warfarin dosage. Demographic characteristics, concomitant medications, daily dosage of warfarin, CYP2C9 and VKORC1 genotypes, and INR were recorded. Data analysis based on the Michaelis-Menten equation to describe the relationship between daily warfarin dose and INR was performed using NONMEM. The reliability and stability of the final model were evaluated using goodness-of-fit plots, resampling techniques with a nonparametric bootstrap, and external data. RESULTS: The daily warfarin dose and INR were described by a more pharmacologically expressive model than multivariate linear regression (MLR) model. The population standard value of Km was 3.56 mg, and the Hill coefficient was 0.512, with individual variabilities of 53.1% and 55.9%, respectively. CYP2C9 *1/*3, VKORC1 AA, concomitant amiodarone, and nonheart valve replacement reduced the warfarin Km by 30.4%, 74.3%, 34.5%, and 39.4%, respectively. The Km value decreased with age and increased with fat free mass (FFM). INR prediction error (73.0%) of the external datasets was within ± 20%. CONCLUSION: A dose-response model of warfarin was established based on the relationship between daily warfarin dose and INR. Expected genotype effects on Km and demographic characteristics were confirmed. The model has the potential to be a powerful tool for individualized warfarin therapy for Chinese outpatients.

[Correlation analysis between the numbers of G-MDSCs and M-MDSCs in peripheral blood decrease and therapeutic effect in patients with colorectal cancer].

Objective To detect the frequencies of myeloid-derived suppressor cells (MDSCs) in the peripheral blood of patients with colorectal cancer (CRC) in an attempt to investigate the relationship between the change of MDSC frequencies and clinicopathologic characteristics of CRC patients. Methods The frequencies of granulocytic MDSCs (G-MDSCs) and monocytic MDSCs (M-MDSCs) in the peripheral blood of 82 CRC patients and 30 healthy volunteers were detected by flow cytometry. ANOVA and t-test were used to explore the relationship between the frequencies of MDSCs in the peripheral blood and the TNM stages, lymph node metastasis, tumor sites, histological grade as well as common therapeutic measures of CRC patients. Correlation analysis was performed to evaluate the correlation between the frequencies of G-MDSCs and M-MDSCs. Results Compared with the 30 healthy volunteers, the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of the 82 CRC patients were statistically higher. The frequencies of G-MDSCs and M-MDSCs in the peripheral blood from TNF stage III and IV CRC patients were obviously higher than those from stage I and II patients, and CRC patients with lymph node metastasis had remarkably higher frequencies of G-MDSCs and M-MDSCs than the patients without lymph node metastasis. There were no statistical differences in the frequencies of G-MDSCs and M-MDSCs in the peripheral blood between CRC patients with different tumor sites as well as histological grades. Radical resection and effective adjuvant chemotherapy significantly decreased the frequencies of G-MDSCs and M-MDSCs in the peripheral blood of CRC patients. No statistical correlation was found between the frequencies of circulating G-MDSCs and M-MDSCs in CRC patients. Conclusion G-MDSC and M-MDSC frequencies in the peripheral blood of CRC patients significantly increased, and were closely associated with TNM stages and lymph node metastasis. Radical resection and effective adjuvant chemotherapy significantly reduced the frequencies of circulating G-MDSCs sand M-MDSCs of CRC patients.

[Expression of COX-1 and COX-2 in the Platelet of Iron Deficiency Anemia Women at Childbearing Age and Its Clinical Significance].

OBJECTIVE: To detect the expression level of cyclooxygenase-1（COX-1） and cyclooxygenase-2（COX-2） in the platelet of iron deficiency anemia（IDA）women at childbearing age and to explore its correlation with the different indexes of anemia and platelets. METHODS: Forty female IDA patients at childbearing age and 35 healthy controls were enrolled in this study. The Flow cytometry was used to detect the expression of platelet COX-1 and COX-2，the platelet aggregation function as examined by turbidimetric method，and the levels of serum ferritin were analyzed by electrochemical luminescence method，the leval of serum iron was determined by ELISA，and the correlation of different indexes was analyzed. RESULTS: Compared with healthy controls，the levels of platelet COX-1 and COX-2 were significantly lower in female IDA patients at Childbearing age（P<0.05），but platelet count（Plt），mean platelet volume（MPV） and platelet aggregation rate（PAgT）were not statistically different between the 2 groups（P > 0.05）. The expression level of platelet COX-1 positively correlated with those of Hb（r =0.623，P<0.01），serum iron（r =0.321，P<0.05） and HCT（r=0.305，P<0.05）. but the platelet COX-2 expression did not corelate with these indexs. CONCLUSION: The expression of platelet COX-1 and COX-2 in female IDA patients at Childbearing age markedly decrease，and the expression level of platelet COX-1 closely relates with the severity of anemia，that possesses reference value for clinical diagnosis of female IDA patients at Childbearing age..

miR-1179 promotes cell invasion through SLIT2/ROBO1 axis in esophageal squamous cell carcinoma.

MiR-1179, a new identified miRNA highly associated with metastasis of colorectal cancer which was never reported in esophageal squamous cell carcinoma (ESCC). Here we measured the expression levels of miR-1179 and the candidate target gene in tissues from 40 patients with ESCC. Transwell, Dual-luciferase reporter assay and immunocytochemistry assay were employed to detect the function role of miR-1179 in vitro. We found that miR-1179 was up-regulated in human ESCC tumor tissues. Bioinformatics analysis indicated that SLIT2 acting as a new potential target of miR-1179 which was confirmed by luciferase reporter assay. Down-regulation of miR-1179 suppressed cell invasion in vitro with an increasing level of SLIT2 and ROBO1, besides, the up-regulation of SLIT2 decreased cell invasion through ROBO1. Taken together, these findings will shed light the role to mechanism of miR-1179 in regulating cell invasion via SLIT2/ROBO1 axis.

miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5.

MicroRNAs (miRNAs) are short, non-coding RNAs (∼ 22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating numerous target genes at posttranscriptional level. However, the role of microRNAs in lung cancer, particularly non-small-cell lung cancer (NSCLC), has remained elusive. In this study, two microRNAs, miR-1271 and miR-628, and their predicted target genes were identified differentially expressed in NSCLC by analyzing the miRNA and mRNA expression data from NSCLC tissues and their matching normal controls. miR-1271 and its target gene HOXA5 were selected for further investigation. CCK-8 proliferation assay showed that the cell proliferation was promoted by miR-1271 in NSCLC cells, while miR-1271 inhibitor could significantly inhibited the proliferation of NSCLC cells. Interestingly, migration and invasion assay indicated that overexpression of miR-1271 could significantly promoted the migration and invasion of NSCLC cells, whereas miR-1271 inhibitor could inhibited both cell migration and invasion of NSCLC cells. Western blot showed that miR-1271 suppressed the protein level of HOXA5, and luciferase assays confirmed that miR-1271 directly bound to the 3'untranslated region of HOXA5. This study indicated indicate that miR-1271 regulates NSCLC cell proliferation and invasion, via the down-regulation of HOXA5. Thus, miR-1271 may represent a potential therapeutic target for NSCLC intervention.

[Correlation analysis between killer cell immunoglobulin-like receptor gene polymorphism and primary biliary cirrhosis].

OBJECTIVE: To explore the association between killer cell immunoglobulin-like receptor (KIR) gene polymorphism and primary biliary cirrhosis (PBC) in Jiangsu Han population. METHODS: Peripheral blood samples were taken from 122 patients with PBC hospitalized in third people's hospital of Changzhou in Jiangsu province between June 2011 and July 2013 and 198 randomly matched unrelated healthy donors. KIR was typed by SYBR(R)Green I real-time PCR genotyping assay. Gene and genotype frequencies of KIR were compared between the two groups. RESULTS: The gene frequencies of KIR2DL2 and 2DS2 were significantly lower in the PBC group as compared with those of the control group. KIR2DL2 and 2DS2 were associated with lower risk of PBC. The total 55 KIR genotypes were found in the patients (24 genotypes) and the controls (47 genotypes). The most commonly observed KIR genotype was AA1 , and the next were BX2, BX8, BX4 and BX14 . Only 11 genotypes were observed in five individuals or more, allowing for a meaningful comparison of genotype frequencies between the patients and controls. There was no statistically significant difference in genotype frequencies between the two groups. CONCLUSION: The absence of KIR2DL2/2DS2 genotype may be associated with protection against PBC in Jiangsu Han population.

Interrelationships between ALOX5AP polymorphisms, serum leukotriene B4 level and risk of acute coronary syndrome.

BACKGROUND: We investigated the relationships between the ALOX5AP gene rs10507391 and rs4769874 polymorphisms, serum levels of leukotriene (LT) B4, and risk of acute coronary syndrome (ACS). METHODS: A total of 709 participants, comprising 508 ACS patients (ACS group) and 201 noncoronary artery disease patients with chest pain (control group) were recruited from the Han population of the Changwu region in China. Two polymorphic loci were genotyped using polymerase chain reaction and restriction fragment length polymorphism analysis. Serum LTB4 level was determined by enzyme-linked immunosorbent assay. RESULTS: Serum LTB4 levels were significantly higher (P<0.001) in the ACS group (median/interquartile range, 470.27/316.32 pg/ml) than in the control group (233.05/226.82 pg/ml). No statistical differences were observed between genotype, allele and haplotype frequencies for the tested loci in either the ACS group or the control group, even after adjustments were made for conventional risk factors by multivariate logistic regression. This suggests there is no association between the ALOX5AP rs10507391 and rs4769874 polymorphisms and ACS risk. Elevated serum LTB4 level was closely linked to ACS risk, and may be independent of traditional risk factors as a risk factor for ACS (P<0.001). There was no significant association between serum LTB4 levels and the two variants in either the ACS group or the control group. CONCLUSIONS: Rs10507391, rs4769874 and its haplotypes in ALOX5AP are unrelated to ACS risk in the Chinese Han population of Changwu, but elevated serum LTB4 level is strongly associated with ACS risk. Serum LTB4 level is not subject to the influence of either the rs10507391, rs4769874 or the haplotype.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers, part I: core region.

Ritonavir (RTV), an HIV-1 protease inhibitor (PI), is also a potent mechanism-based inhibitor of human cytochrome P450 3A (CYP3A) and has been widely prescribed as a pharmacoenhancer. As a boosting agent for marketed PIs, it reduces pill burden, and improves compliance. Removal of the hydroxyl group from RTV reduces, but does not eliminate HIV PI activity and does not affect CYP3A inhibition. Herein we report the discovery of a novel series of CYP3A inhibitors that are devoid of antiviral activity. The synthesis and evaluation of analogs with extensive modifications of the 1,4-diamine core along with the structure activity relationships with respect to anti-HIV activity, CYP3A inhibitory activity, selectivity against other CYP enzymes and the human pregnane X receptor (PXR) will be discussed.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers. Part II: P2/P3 region and discovery of cobicistat (GS-9350).

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.

Discovery of ledipasvir (GS-5885): a potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection.

A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an extended plasma half-life of 37-45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. 39 has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.

Discovery of GS-9669, a thumb site II non-nucleoside inhibitor of NS5B for the treatment of genotype 1 chronic hepatitis C infection.

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges.

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.

Synthesis and biological evaluation of phosphonate analogues of nevirapine.

A series of nevirapine-based analogues containing the phosphonate functionality were prepared and evaluated in vitro against HIV RT. The effect of the phosphonate was evaluated against the wild type and Y181C HIV replication. An in vivo PK study was performed on a select analogue.