Development of Cilofexor, an intestinally-biased Farnesoid X Receptor agonist, for the treatment of fatty liver disease.

The farnesoid X receptor (FXR) is a nuclear receptor that controls bile acid, lipid, and cholesterol metabolism. FXR-targeted drugs have shown promise in late-stage clinical trials for non-alcoholic steatohepatitis. Herein, we used clinical results from our first non-steroidal FXR agonist, Px-102 (4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl] benzoic acid), to develop cilofexor, a potent, non-steroidal FXR agonist with a more manageable safety profile. Px-102 demonstrated the anticipated pharmacodynamic (PD) effects in healthy volunteers but caused a 2-fold increase in alanine aminotransferase (ALT) activity and changes in cholesterol levels. These data guided development of a high fat diet mouse model to screen FXR agonists based on ALT and cholesterol changes. Cilofexor was identified to elicit only minor changes in these parameters. The differing effects of cilofexor and Px-102 on ALT/cholesterol in the model could not be explained by potency or specificity, and we hypothesized that the relative contribution of intestinal and liver FXR activation may be responsible. Gene expression analysis from rodent studies revealed that cilofexor, but not Px-102, had a bias for FXR transcriptional activity in the intestine compared to the liver. Fluorescent imaging in hepatoma cells demonstrated similar subcellular localization for cilofexor and Px-102, but cilofexor was more rapidly washed out, consistent with a lower membrane residence time contributing to reduced hepatic transcriptional effects. Cilofexor demonstrated antisteatotic and antifibrotic efficacy in rodent models and antisteatotic efficacy in a monkey model, with the anticipated PD and a manageable safety profile in human phase I studies. Significance Statement FXR (farnesoid X receptor) agonists have shown promise in treating non-alcoholic steatohepatitis and other liver diseases in the clinic, but balancing efficacy with undesired side effects has been difficult. Here, we examined the preclinical and clinical effects of the first-generation FXR agonist, Px-102 (4-[2-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-4-isoxazolyl]methoxy]phenyl]cyclopropyl] benzoic acid), to enable the selection of an analog, cilofexor, with unique properties that reduced side effects yet maintained efficacy. Cilofexor is one of few remaining FXR agonists in clinical development.

Comparison of stillbirth trends over two decades in Wales, United Kingdom and Western Australia: An international retrospective cohort study.

BACKGROUND: Stillbirth is a critical public health issue worldwide. While the rates in high-income countries are relatively low, there are persistent between-country disparities. OBJECTIVES: To compare stillbirth rates and trends in Wales and the State of Western Australia (WA), Australia, and provide insights into any differences. METHODS: In this international retrospective cohort study, we pooled population-based data collections of all births ≥24 weeks' gestation (excluding terminations for congenital anomalies) between 1993 and 2015, divided into six time periods. The stillbirth rate per 1000 births was estimated for each cohort in each time period. Multivariable Poisson regression analyses, adjusted for appropriateness of growth, socio-economic status, maternal age, and multiple birth, were performed to evaluate the interaction between cohort and time period. Relative risk (RR) and 95% confidence interval (CI) for each time period and cohort were calculated. RESULTS: There were 767 731 births (3725 stillbirths) in Wales and 648 373 (2431 stillbirths) in WA. The overall stillbirth rate declined by 15.9% over the study period in Wales (from 5.3 in 1993-96 to 4.5 per 1000 births in 2013-15; Ptrend  < .01) but by 40.4% in WA (from 4.9 to 2.9 per 1000 births in WA; Ptrend  < .01). Using 1993-96 in WA as the reference group, the adjusted RRs for stillbirths at 37-38 weeks' gestation in the most recent study period (2013-15) were 0.85 (95% CI 0.64, 1.13) in Wales and 0.51 (95% CI 0.36, 0.73) in WA. CONCLUSIONS: The stillbirth rates between Wales and WA have widened in the last two decades (especially among late-term births), although the absolute rates for both are distinctly higher than the best-performing nations. While the differences may be partly explained by timing of birth and maternal life style behaviours such as smoking, it is important to identify and ameliorate the associated risk factors to support a reduction in preventable stillbirths.

Selectivity switch between FAK and Pyk2: Macrocyclization of FAK inhibitors improves Pyk2 potency.

Herein, we describe the synthesis of Pyk2 inhibitors via macrocyclization of FAK and dual Pyk2-FAK inhibitors. We identified macrocycle 25a as a highly potent Pyk2 inhibitor (IC50=0.7nM), with ∼175-fold improvement in Pyk2 potency as compared to its acyclic counterpart. In many cases, macrocyclization improved Pyk2 potency while weakening FAK potency, thereby improving the Pyk2/FAK selectivity ratio for this structural class of inhibitors. Various macrocyclic linkers were studied in an attempt to optimize Pyk2 selectivity. We observed macrocyclic atropisomerism during the synthesis of 19-membered macrocycles 10a-d, and successfully obtained crystallographic evidence of one atropisomer (10a-AtropB) preferentially bound to Pyk2.

Preclinical characterization of GS-9669, a thumb site II inhibitor of the hepatitis C virus NS5B polymerase.

GS-9669 is a highly optimized thumb site II nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, with a binding affinity of 1.35 nM for the genotype (GT) 1b protein. It is a selective inhibitor of HCV RNA replication, with a mean 50% effective concentration (EC(50)) of ≤ 11 nM in genotype 1 and 5 replicon assays, but lacks useful activity against genotypes 2 to 4. The M423T mutation is readily generated clinically upon monotherapy with the thumb site II inhibitors filibuvir and lomibuvir, and it is notable that GS-9669 exhibited only a 3-fold loss in potency against this variant in the genotype 1b replicon. Rather than M423T, resistance predominantly tracks to residues R422K and L419M and residue I482L in GT 1b and 1a replicons, respectively. GS-9669 exhibited at least additive activity in combination with agents encompassing four other direct modes of action (NS3 protease, NS5A, NS5B via an alternative allosteric binding site, and NS5B nucleotide) as well as with alpha interferon or ribavirin in replicon assays. It exhibited high metabolic stability in in vitro human liver microsomal assays, which, in combination with its pharmacokinetic profiles in rat, dog, and two monkey species, is predictive of good human pharmacokinetics. GS-9669 is well suited for combination with other orally active, direct-acting antiviral agents in the treatment of genotype 1 chronic HCV infection. (This study has been registered at ClinicalTrials.gov under registration number NCT01431898.).

Exercise-induced bronchoconstriction in school-aged children who had chronic lung disease in infancy.

OBJECTIVES: To assess for exercise-induced bronchoconstriction in 8- to 12-year-old children who had chronic lung disease (CLD) in infancy, and to evaluate the response of bronchoconstriction to bronchodilation with albuterol in comparison with preterm and term controls. STUDY DESIGN: Ninety-two children, including 29 with CLD, 33 born preterm at ≤32 weeks' gestation, and 30 born at term, underwent lung spirometry before and after cycle ergometry testing and after postexercise bronchodilation with albuterol. RESULTS: Doctor-diagnosed asthma and exercise-induced wheeze were reported more frequently in the CLD group than in the preterm and term groups, but only 10% were receiving a bronchodilator. There were no differences among the groups in peak minute ventilation, oxygen uptake, or carbon dioxide output at maximum exercise. After maximal exercise, predicted forced expiratory volume in 1 second (FEV1) decreased from a mean baseline value of 81.9% (95% CI, 76.6-87.0%) to 70.8% (95% CI, 65.5-76.1%) after exercise in the CLD group, from 92.0% (95% CI, 87.2-96.8%) to 84.3% (95% CI, 79.1-89.4%) in the preterm group, and from 97.5% (95% CI, 92.5-102.6%) to 90.3% (95% CI, 85.1-95.5%) in the term group. After albuterol administration, FEV1 increased to 86.8% (95% CI, 81.7-92.0%) in the CLD group, 92.1% (95% CI, 87.3-96.9%) in the preterm group, and 97.1% (95% CI, 92.0-102.3%) in the term group. The decrease in predicted FEV1 after exercise and increase in predicted FEV1 after bronchodilator use were greatest in the CLD group (-11.0% [95% CI, -18.4 to -3.6%] and 16.0% [95% CI, 8.6-23.4%], respectively; P < .005 for both), with differences of <8% in the 2 control groups. CONCLUSION: School-age children who had CLD in infancy had significant exercise-induced bronchoconstriction that responded significantly to bronchodilation. Reversible exercise-induced bronchoconstriction is common in children who experienced CLD in infancy and should be actively assessed for and treated.

Mechanism for effective lymphoid cell and tissue loading following oral administration of nucleotide prodrug GS-7340.

GS-7340 is a prodrug of tenofovir (TFV) that more efficiently delivers TFV into lymphoid cells and tissues than the clinically used prodrug TFV disoproxil fumarate, resulting in higher antiviral potency at greatly reduced doses and lower systemic TFV exposure. First-pass extraction by the intestine and liver represents substantial barriers to the oral delivery of prodrugs designed for rapid intracellular hydrolysis. In order to understand how GS-7340 reduces first-pass clearance to be an effective oral prodrug, its permeability and stability were characterized in vitro and detailed pharmacokinetic studies were completed in dogs. GS-7340 showed concentration-dependent permeability through monolayers of caco-2 cells and dose-dependent oral bioavailability in dogs, increasing from 1.7% at 2 mg/kg to 24.7% at 20 mg/kg, suggesting saturable intestinal efflux transport. Taking into account a 65% hepatic extraction measured in portal vein cannulated dogs, high dose GS-7340 is nearly completely absorbed. Consistent with the proposed role of intestinal efflux transport, coadministration of low dose GS-7340 with a transport inhibitor substantially increased GS-7340 exposure. The result of effective oral absorption and efficient lymphoid cell loading was reflected in the high and persistent levels of the pharmacologically active metabolite, TFV diphosphate, in peripheral blood mononuclear cells following oral administration to dogs. In conclusion, GS-7340 reaches the systemic circulation to effectively load target cells by saturating intestinal efflux transporters, facilitated by its high solubility, and by maintaining sufficient stability in intestinal and hepatic tissue.

HCV versus HIV drug discovery: Déjà vu all over again?

Efforts to address HIV infection have been highly successful, enabling chronic suppression of viral replication with once-daily regimens. More recent research into HCV therapeutics have also resulted in very promising clinical candidates. This Digest explores similarities and differences in the two fields and compares the chronology of drug discovery relative to the availability of enabling tools, and concludes that safe and convenient, once-daily regimens are likely to reach approval much more rapidly for HCV than was the case for HIV.

Selective modulation of hepatic cytochrome P450 and flavin monooxygenase 3 expression during citrobacter rodentium infection in severe combined immune-deficient mice.

The profile of selective modulation of hepatic cytochrome P450 (P450) gene expression caused by infection with the murine intestinal pathogen Citrobacter rodentium has been well characterized in multiple genetic backgrounds; yet, the mechanisms underlying this modulation are still not entirely understood. Although several studies have addressed the roles of cytokines from the innate immune system, the influence of the adaptive immune system is not known. To address this deficiency, we used mice harboring the severe combined immune deficiency (SCID) spontaneous mutation, which lack mature T and B lymphocytes and are unable to mount an acquired immune response. Female C57BL/6 (B6) and SCID mice were infected orally with C. rodentium and assessed for bacterial colonization/translocation and P450 and flavin monooxygenase-3 (Fmo3) expression levels after 7 days. SCID mice showed similar patterns of colonic bacterial colonization and a similar degree of colonic mucosal hypertrophy compared with infected B6 mice, but SCID mice displayed 6-fold greater bacterial translocation to the liver. In the SCID mice, Cyp4a10 and Cyp2b9 down-regulations were partially and fully blocked, respectively, whereas the regulation of other P450s and Fmo3 was similar in both strains. In the C3H genetic background, the SCID mutation also blocked the down-regulation of Cyp3a11, Cyp3a25, Cyp2d22, and Cyp2c29. The results clearly dissociate bacterial translocation to the liver from hepatic drug-metabolizing enzyme regulation and suggest a possible role of T cells, T-cell cytokines, or other proteins regulated by such cytokines in the selective regulation of a limited subset of hepatic P450 enzymes during C. rodentium infection.

Tri-substituted acylhydrazines as tertiary amide bioisosteres: HCV NS5B polymerase inhibitors.

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.

Estimating insertion length of umbilical arterial and venous catheters in newborn infants: time for change.

BACKGROUND: Umbilical catheters are inserted through the umbilical artery or vein at birth and are crucial in neonatal care. There are several different methods of estimating adequate insertion length of umbilical catheters based on one of two hypotheses; that the insertion length of the UC is correlated to either the infant's birth weight or an external length measurement. AIM: To review the published literature on methods of estimating insertion lengths of umbilical arterial catheters (UACs) and umbilical venous catheters (UVCs) in newborn infants. METHODS: Systematic search on Medline was undertaken using keywords for relevant papers up to March 2019. Papers were selected by manual search of titles and abstracts. RESULTS: Formulae for predicting umbilical catheter insertion length are unreliable, particularly for UVCs. There is also conflicting evidence around whether birth weight-based formulae are more reliable than external length-based formulae. Studies comparing various methods to determine their efficacy to show that current formulae have a low accuracy for determining both UVC and UAC positioning. CONCLUSIONS: Current formulae for estimating insertion length of umbilical catheters are not fit for purpose. We propose a new observational study which uses a new external length measurement, the sternal notch to umbilicus length, to develop a more reliable formula for the insertion of UVC and UAC to an adequate length.

Thymoglobulin Versus Alemtuzumab Versus Basiliximab Kidney Transplantation From Donors After Circulatory Death.

Introduction: The Campath, Calcineurin inhibitor (CNI) reduction, and Chronic allograft nephropathy (3C), a study comparing alemtuzumab versus basiliximab induction immunosuppression in kidney transplants, has found lower acute rejection rate with alemtuzumab but same graft survival. The aim of the current study is to evaluate the effect of induction immunosuppression (thymoglobulin, alemtuzumab, basiliximab) on the outcome of kidneys of donors after circulatory death (DCD). Methods: Data of the 274 DCD patients of the 3C obtained from the sponsor were compounded with the 140 DCD patients who received thymoglobulin in a single center with the same entry criteria as the 3C, giving 414 patients on 3 induction regimes. Results: There were more male donors (P < 0.05) and human leukocyte antigen and DR mismatched patients in the thymoglobulin group (P < 0.001). Death-censored graft survival at 6 months was 98.6% in the thymoglobulin, 95.5% in the alemtuzumab (P = 0.08), and 95.7% in the basiliximab group (P = 0.09) and at 2 years 97.9% versus 94.8% (P = 0.13, hazard ratio [HR] 2.8, 95% CI 0.7-10.9) versus 94.3% (P = 0.06, HR 3.5, 95% CI 0.9-13.6), respectively.The 2-year overall graft survival was 95% in the thymoglobulin versus 88% in the alemtuzumab (unadjusted P = 0.038, adjusted HR 2.4, 95% CI 0.99-5.9) and 91.4% in the basiliximab group (P = 0.21). The 2-year patient survival was numerically less in the alemtuzumab compared with the thymoglobulin group (91.8% vs. 97.1%, P = 0.052, HR 2.90, 95% CI 0.93-9.2). Acute rejection was 17% in the basiliximab, 4.3% in the thymoglobulin, and 6% in the alemtuzumab group (P < 0.001). Conclusion: In DCD transplants, thymoglobulin induction may provide advantage over alemtuzumab in patient survival and the same advantage as alemtuzumab over basiliximab in terms of acute rejection. Differing maintenance immunosuppression may contribute to the difference found.

Neonatal sepsis and mortality in low-income and middle-income countries from a facility-based birth cohort: an international multisite prospective observational study.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

Structural and binding analysis of pyrimidinol carboxylic acid and N-hydroxy quinazolinedione HIV-1 RNase H inhibitors.

HIV-1 RNase H breaks down the intermediate RNA-DNA hybrids during reverse transcription, requiring two divalent metal ions for activity. Pyrimidinol carboxylic acid and N-hydroxy quinazolinedione inhibitors were designed to coordinate the two metal ions in the active site of RNase H. High-resolution (1.4 Å to 2.1 Å) crystal structures were determined with the isolated RNase H domain and reverse transcriptase (RT), which permit accurate assessment of the metal and water environment at the active site. The geometry of the metal coordination suggests that the inhibitors mimic a substrate state prior to phosphodiester catalysis. Surface plasmon resonance studies confirm metal-dependent binding to RNase H and demonstrate that the inhibitors do not bind at the polymerase active site of RT. Additional evaluation of the RNase H site reveals an open protein surface with few additional interactions to optimize active-site inhibitors.

miR-141 mediates recovery from acute kidney injury.

Acute kidney injury (AKI) is a global clinical problem characterised by a sudden decline in renal function and mortality as high as 60%. Current AKI biomarkers have limited ability to classify disease progression and identify underlying pathological mechanisms. Here we hypothesised that alterations in urinary microRNA profiles could predict AKI recovery/nonrecovery after 90 days, and that injury-specific changes would signify microRNA mediators of AKI pathology. Comparison of urinary microRNA profiles from AKI patients with controls detected significant injury-specific increases in miR-21, miR-126 and miR-141 (p < 0.05) and decreases in miR-192 (p < 0.001) and miR-204 (p < 0.05). Expression of miR-141 increased in renal proximal tubular epithelial cells (PTECs) under oxidative stress in vitro and unilateral ischaemic reperfusion injury in vivo. Forced miR-141 expression in the presence of H2O2 increased PTEC death and decreased cell viability. Of nine messenger RNA targets with two or more miR-141 3'-untranslated region binding sites, we confirmed protein tyrosine phosphatase receptor type G (PTPRG) as a direct miR-141 target in PTECs. PTPRG-specific siRNA knockdown under oxidative stress increased PTEC death and decreased cell viability. In conclusion, we detected significant alterations in five urinary microRNAs following AKI, and identified proximal tubular cell PTPRG as a putative novel therapeutic target.

A role for arthropods as vectors of multidrug-resistant Enterobacterales in surgical site infections from South Asia.

Understanding how multidrug-resistant Enterobacterales (MDRE) are transmitted in low- and middle-income countries (LMICs) is critical for implementing robust policies to curb the increasing burden of antimicrobial resistance (AMR). Here, we analysed samples from surgical site infections (SSIs), hospital surfaces (HSs) and arthropods (summer and winter 2016) to investigate the incidence and transmission of MDRE in a public hospital in Pakistan. We investigated Enterobacterales containing resistance genes (blaCTX-M-15, blaNDM and blaOXA-48-like) for identification, antimicrobial susceptibility testing and whole-genome sequencing. Genotypes, phylogenetic relationships and transmission events for isolates from different sources were investigated using single-nucleotide polymorphism (SNP) analysis with a cut-off of ≤20 SNPs. Escherichia coli (14.3%), Klebsiella pneumoniae (10.9%) and Enterobacter cloacae (16.3%) were the main MDRE species isolated. The carbapenemase gene blaNDM was most commonly detected, with 15.5%, 15.1% and 13.3% of samples positive in SSIs, HSs and arthropods, respectively. SNP (≤20) and spatiotemporal analysis revealed linkages in bacteria between SSIs, HSs and arthropods supporting the One Health approach to underpin infection control policies across LMICs and control AMR.

The Non-Steroidal FXR Agonist Cilofexor Improves Portal Hypertension and Reduces Hepatic Fibrosis in a Rat NASH Model.

BACKGROUND: The farnesoid X receptor (FXR) influences hepatic metabolism, inflammation and liver fibrosis as key components of non-alcoholic steatohepatitis (NASH). We studied the effects of the non-steroidal FXR agonist cilofexor (formerly GS-9674) on portal pressure and fibrosis in experimental NASH. METHODS: NASH was induced in Wistar rats using a choline-deficient high-fat diet plus intraperitoneal sodium nitrite injections. First, a dose-finding study was performed with 10 mg/kg and 30 mg/kg of cilofexor, focusing on histological readouts. Liver fibrosis was assessed by Picro-Sirius-Red, desmin staining and hepatic hydroxyproline content. Gene expression was determined by RT-PCR. In a subsequent hemodynamic study, rats received 30 mg/kg cilofexor with or without propranolol (25 mg/kg). Portal pressure, systemic hemodynamics and splanchnic blood flow were measured. RESULTS: Cilofexor dose-dependently induced FXR target genes shp, cyp7a1 and fgf15 in hepatic and ileal tissues, paralleled by a dose-dependent reduction in liver fibrosis area (Picro-Sirius-Red) of -41% (10 mg/kg) and -69% (30 mg/kg), respectively. The 30 mg/kg cilofexor dose significantly reduced hepatic hydroxyproline content (-41%), expression of col1a1 (-37%) and pdgfr-ß (-36%), as well as desmin area (-42%) in NASH rats. Importantly, cilofexor decreased portal pressure (11.9 ± 2.1 vs. 8.9 ± 2.2 mmHg; p = 0.020) without affecting splanchnic blood-flow or systemic hemodynamics. The addition of propranolol to cilofexor additionally reduced splanchnic inflow (-28%) but also mean arterial pressure (-25%) and heart rate (-37%). CONCLUSION: The non-steroidal FXR agonist cilofexor decreased portal hypertension and reduced liver fibrosis in NASH rats. While cilofexor seems to primarily decrease sinusoidal resistance in cirrhotic portal hypertension, the combination with propranolol additionally reduced mesenteric hyperperfusion.

Assessment of GS-9219 in a pet dog model of non-Hodgkin's lymphoma.

PURPOSE: To assess, in dogs with naturally occurring non-Hodgkin's lymphoma, pharmacokinetics, safety, and activity of GS-9219, a prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG), which delivers PMEG and its phosphorylated metabolites to lymphoid cells with preferential cytotoxicity in cells with a high proliferation index such as lymphoid malignancies. EXPERIMENTAL DESIGN: To generate proof-of-concept, a phase I/II trial was conducted in pet dogs (n = 38) with naturally occurring non-Hodgkin's lymphoma using different dose schedules of GS-9219. A subset of dogs was further evaluated with 3'-deoxy-3'-(18)F-fluorothymidine positron emission tomography/computed tomography imaging before and after treatment. RESULTS: The prodrug had a short plasma half-life but yielded high and prolonged intracellular levels of the cytotoxic metabolite PMEG diphosphate in peripheral blood mononuclear cells in the absence of detectable plasma PMEG. Dose-limiting toxicities were generally manageable and reversible and included dermatopathy, neutropenia, and gastrointestinal signs. Antitumor responses were observed in 79% of dogs and occurred in previously untreated dogs and dogs with chemotherapy-refractory non-Hodgkin's lymphoma. The median remission durations observed compare favorably with other monotherapies in dogs with non-Hodgkin's lymphoma. High 3'-deoxy-3'-(18)F-fluorothymidine uptake noted in lymphoid tissues before treatment decreased significantly after treatment (P = 0.016). CONCLUSIONS: GS-9219 was generally well tolerated and showed significant activity against spontaneous non-Hodgkin's lymphoma as modeled in pet dogs and, as such, supports clinical evaluation in humans.

Waltzing transporters and 'the dance macabre' between humans and bacteria.

Multidrug-resistance efflux pumps - in particular those belonging to the resistance-nodulation-cell-division (RND) family of transporters, with their unusually high degree of substrate promiscuity - significantly restrict the effectiveness of antibacterial therapy. Recent years have heralded remarkable insights into the structure and mechanisms of these fascinating molecular machines. Here, we review recent advances in the field and describe various approaches used in combating efflux-mediated resistance.

Immune Remodeling of the Extracellular Matrix Drives Loss of Cancer Stem Cells and Tumor Rejection.

The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.

Metabolic Dysregulation of the Lysophospholipid/Autotaxin Axis in the Chromosome 9p21 Gene SNP rs10757274.

BACKGROUND: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA. METHODS: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes. RESULTS: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were DGKA, MBOAT2, PLPP1, and LPL. The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p. CONCLUSIONS: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.