Phase I Study of GS-3583, an FMS-like Tyrosine Kinase 3 Agonist Fc Fusion Protein, in Patients with Advanced Solid Tumors.

PURPOSE: GS-3583, an FMS-like tyrosine kinase 3 (FLT3) agonist Fc fusion protein, expanded conventional dendritic cells (cDC) in the periphery of healthy volunteers, suggesting potential for GS-3583 to increase cDCs in the tumor microenvironment and promote T cell-mediated antitumor activity in cancer patients. This phase Ib open-label study assessed GS-3583 in adults with advanced solid tumors. PATIENTS AND METHODS: Multiple escalating doses of GS-3583 (standard 3+3 design) were administered intravenously on days 1 and 15 of cycle 1 and day 1 of each subsequent 28-day cycle for up to 52 weeks. Dose-limiting toxicity (DLT) was evaluated during the first 28 days of GS-3583 at each dose level. RESULTS: Thirteen participants enrolled in four dose-escalation cohorts, after which the study was terminated following safety review. Median (range) age was 71 (44-79), and 7 (54%) participants were male. There were no DLTs. Seven participants had grade ≥3 AEs; 2 participants had grade 5 AEs, including a second primary malignancy (acute myeloid leukemia) considered treatment-related. Dose-dependent increase in GS-3583 serum exposure was observed in the dose range of 2-20 mg with GS-3583 accumulation at higher dose levels. Expansions of cDCs occurred at all four doses with a dose-dependent trend in the durability of the cDC expansion. CONCLUSIONS: GS-3583 was relatively well tolerated and induced dose-dependent expansion of cDCs in the periphery of patients with advanced solid tumors. However, development of a second primary malignancy provides a cautionary tale for the FLT3 agonist mechanism. See related commentary by Raeder and Drazer, p. 2857.

Biosynthesis, Characterization, Evaluation, and Shelf-Life Study of Silver Nanoparticles against Cotton Bollworm, Helicoverpa armigera (Hubner) (Noctuidae: Lepidoptera).

Nanoparticles provide a promising and alternative platform of eco-friendly technologies that encompasses better cost-resilient remedies against one of the most economically harnessing insect pests of cotton. The main goal of this research was to provide a better management strategy through biologically synthesizing (sunlight exposure method) green nanoparticles from leaf extracts of Azadirachta indica and Pongamia pinnata and proving their bioefficacy on H. armigera (2nd instar). Characterization of bio-synthesized silver nanoparticles was carried out using UV-Visible spectroscopy for confirming the formation of nanoparticles, a Particle Size Analyzer (PSA) for determining the size/distribution of particles, and a Scanning Electron Microscope (SEM) for analyzing the surface topology of nanoparticles. The results obtained from PSA analysis showed that A. indica and P. pinnata-based silver nanoparticles had an average diameter of 61.70 nm and 68.80, respectively. Topographical images obtained from SEM proved that most of the green synthesized silver nanoparticles were spherical in shape. A. indica-based silver nanoparticles were found to be comparatively more efficient and have higher insecticidal activity compared to P. pinnata-based nanoparticles. A. indica-based AgNPs recorded larval mortality of 60.00 to 93.33 percent at the concentrations of 500 to 2000 ppm, followed by P. pinnata-based nanoparticles, with 60.00 to 90.00 percent larval mortality. Shelf-life studies revealed that A. indica-based AgNPs had the maximum negative zeta potential of -58.96 mV and could be stored for three months without losing bioefficacy and up to six months with negligible reduction in bioefficacy. Symptoms caused by silver nanoparticles were leakage of body fluids, sluggishness, inactiveness, brittleness, etc.

Approaches to handling missing or "problematic" pharmacology data: Pharmacokinetics.

Missing or erroneous information is a common problem in the analysis of pharmacokinetic (PK) data. This may present as missing or inaccurate dose level or dose time, drug concentrations below the analytical limit of quantification, missing sample times, or missing or incorrect covariate information. Several methods to handle problematic data have been evaluated, although no single, broad set of recommendations for commonly occurring errors has been published. In this tutorial, we review the existing literature and present the results of our simulation studies that evaluated common methods to handle known data errors to bridge the remaining gaps and expand on the existing knowledge. This tutorial is intended for any scientist analyzing a PK data set with missing or apparently erroneous data. The approaches described herein may also be useful for the analysis of nonclinical PK data.

Overcoming resistance to anabolic SARM therapy in experimental cancer cachexia with an HDAC inhibitor.

No approved therapy exists for cancer-associated cachexia. The colon-26 mouse model of cancer cachexia mimics recent late-stage clinical failures of anabolic anti-cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx-024. The histone deacetylase inhibitor (HDACi) AR-42 exhibited anti-cachectic activity in this model. We explored combined SARM/AR-42 therapy as an improved anti-cachectic treatment paradigm. A reduced dose of AR-42 provided limited anti-cachectic benefits, but, in combination with GTx-024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR-42 suppressed the IL-6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx-024-mediated ß-catenin target gene regulation was apparent in cachectic mice only when combined with AR-42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx-024 therapy and a blockade of GTx-024-mediated anabolic signaling. AR-42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx-024. Combining GTx-024, a clinically established anabolic therapy, with AR-42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.

Transcriptionally Active Androgen Receptor Splice Variants Promote Hepatocellular Carcinoma Progression.

Owing to the marked sexual dimorphism of hepatocellular carcinoma (HCC), sex hormone receptor signaling has been implicated in numerous aspects of liver cancer pathogenesis. We sought to reconcile the clear contribution of androgen receptor (AR) activity that has been established in preclinical models of HCC with the clinical failure of AR antagonists in patients with advanced HCC by evaluating potential resistance mechanisms to AR-targeted therapy. The AR locus was interrogated for resistance-causing genomic modifications using publicly available primary HCC datasets (1,019 samples). Analysis of HCC tumor and cell line RNA-seq data revealed enriched expression of constitutively active, treatment-refractory AR splice variants (AR-SV). HCC cell lines expressed C-terminal-truncated AR-SV; 28 primary HCC samples abundantly expressed AR-SV. Low molecular weight AR species were nuclear localized and constitutively active. Furthermore, AR/AR-SV signaling promoted AR-mediated HCC cell progression and conferred resistance to AR antagonists. Ligand-dependent and -independent AR signaling mediated HCC epithelial-to-mesenchymal transition by regulating the transcription factor SLUG. These data suggest that AR-SV expression in HCC drives HCC progression and resistance to traditional AR antagonists. Novel therapeutic approaches that successfully target AR-SVs may be therapeutically beneficial for HCC. SIGNIFICANCE: Treatment-refractory, constitutively active androgen receptor splice variants promote hepatocellular carcinoma progression by regulating the epithelial-to-mesenchymal transition pathway.

The adverse effects of interferon-free regimens in 149 816 chronic hepatitis C treated Egyptian patients.

BACKGROUND: Interferon-free regimens are associated with high sustained virological response; however, associated adverse effects have yet to be fully reported. AIM: To evaluate the adverse effects associated with the different direct-acting antiviral drug (DAA) regimens in Egyptian patients. METHODS: This multicenter retrospective study included all adverse effects during and after treatment with DAA regimens of 149 816 chronic hepatitis C treated Egyptian patients. Patients received sofosbuvir (SOF)/ribavirin (RBV) (n = 21 835), SOF/simeprevir (n = 24 215) SOF/daclatasvir (DCV) (n = 58 477), SOF/DCV/RBV (n = 45 188) and paritaprevir/ombitasvir/ritonavir/RBV (n = 101). The duration of treatment varied between 12 and 24 weeks. All changes in the treatment regimens, discontinuation, mortality, and serious side effects were reported. RESULTS: Adverse effects developed in 2475 (1.7%) (mean age [54 ± 9], male gender [53%]) patients. Serious side effects developed in 68% of these patients, and SOF/RBV was the most common causing regimen (73%, P < 0.001). Anaemia and hyperbilirubinemia were the most common side effects (731/149816, 0.5% and 463/149816, 0.3%, respectively) and SOF/RBV (588/21835, 3% and 353/21835, 1.6%, respectively) showed the highest incidence in the treated patients. Hepatocellular carcinoma and mortality were reported in 0.02% and 0.06% of all treated patients, respectively. Patients with liver cirrhosis showed higher incidence of serious side effects (Log rank P = 0.045) and mortality (Log rank P = 0.025) than patients without liver cirrhosis. Male gender (P = 0.012), lower haemoglobin (P < 0.001), platelets (P < 0.001) and albumin (P = 0.001), higher bilirubin (P = 0.002) and cirrhosis (P < 0.001) were factors associated with serious side effects development. CONCLUSION: Adverse effects associated with DAAs are few, anaemia being the most common. SOF/RBV regimen showed the highest rate of side effects while SOF/DCV showed the least.

Role of water chemistry and stabilizers on the Vero-cells-based infectivity of Newcastle disease virus live vaccine.

Newcastle disease virus (NDV) live vaccines are supplied in lyophilized form and usually administered through conventional routes (drinking water, spray, or eye drop) following reconstitution in a diluent. Virus inactivation due to physico-chemical properties of the diluent at the time of administration may lead to vaccine failure. The present study aimed to evaluate the survival of NDV live vaccine strain immersed in 5 pH-amended water samples (pH 5.00, pH 6.00, pH 7.00, pH 8.00, and pH 9.00) by sequential determination of virus infectivity on Vero cells for 3 hours. Minimum reduction in virus infectivity was recorded in the water with neutral or slightly alkaline pH, while the virus was relatively less stable at extreme pH conditions. Maximum reduction of infectivity was observed in the water with pH 9.00 in which the virus was completely inactivated within 3 hours. Addition of stabilizers (Cevamune® or skimmed milk) slightly altered the pH and total dissolved solids (TDS) values of the virus-charged water samples. In the stabilizer-added water samples, minimum reduction in infectivity was observed in the water with neutral pH, followed by the ones with a pH of 8.00, 6.00, 5.00, and 9.00. In all types of water samples, T-90 values (time required for 90% reduction in virus infectivity) were highest (485 minutes) at neutral pH (pH 7.00) and lowest (102 to 134 min) at an extreme alkaline condition (pH 9.00). Results of the present study indicate that water with a pH range of 7.00 to 8.00 is suitable for administration of NDV live vaccines. However, the addition of Cevamune® or skimmed milk may have beneficial effects on preserving the infectivity of the virus, even at extreme pH conditions.

Control of the stereochemistry of C14 hydroxyl during the total synthesis of withanolide E and physachenolide C.

The stereochemical outcome of the epoxidation of Δ14-15 cholestanes with mCPBA is controlled by the steric bulk of a C17 substituent. When the C17 is in the ß configuration, the epoxide is formed in the α face, whereas if the C17 is trigonal (flat) or the substituent is in the α configuration, the epoxide is formed in the ß face. The presence of a hydroxyl substituent at C20 does not influence the stereochemical outcome of the epoxidation.

Hematuria and Hematospermia Associated with the Use of Finasteride for the Treatment of Androgenic Alopecia: A Case Report.

In this report we address an unusual adverse effect of finasteride (Propecia 1 mg tablets) that was associated with painless hematuria and hematospermia in a 38-year-old healthy male during treatment of androgenic alopecia at a dose of 1 mg/day. It was found that the bleeding was linked to finasteride use as it occurred 2-3 days after use and stopped upon discontinuation of the drug. The patient was subjected to urological examination, laboratory investigations, and radiological imaging to identify the probable cause of bleeding. It appeared the bleeding was most probably of prostatic origin in the absence of obvious underlying pathology. The frequency of such unusual bleeding remains to be investigated in large clinical trials to address its exact mechanism, predisposing factors, clinical significance, and potential long-term consequences.

Pharmacogenomics of statins: understanding susceptibility to adverse effects.

Statins are a cornerstone of the pharmacologic treatment and prevention of atherosclerotic cardiovascular disease. Atherosclerotic disease is a predominant cause of mortality and morbidity worldwide. Statins are among the most commonly prescribed classes of medications, and their prescribing indications and target patient populations have been significantly expanded in the official guidelines recently published by the American and European expert panels. Adverse effects of statin pharmacotherapy, however, result in significant cost and morbidity and can lead to nonadherence and discontinuation of therapy. Statin-associated muscle symptoms occur in ~10% of patients on statins and constitute the most commonly reported adverse effect associated with statin pharmacotherapy. Substantial clinical and nonclinical research effort has been dedicated to determining whether genetics can provide meaningful insight regarding an individual patient's risk of statin adverse effects. This contemporary review of the relevant clinical research on polymorphisms in several key genes that affect statin pharmacokinetics (eg, transporters and metabolizing enzymes), statin efficacy (eg, drug targets and pathways), and end-organ toxicity (eg, myopathy pathways) highlights several promising pharmacogenomic candidates. However, SLCO1B1 521C is currently the only clinically relevant pharmacogenetic test regarding statin toxicity, and its relevance is limited to simvastatin myopathy.

GATM polymorphism associated with the risk for statin-induced myopathy does not replicate in case-control analysis of 715 dyslipidemic individuals.

Statin-induced myopathy (SIM) is the most common reason for discontinuation of statin therapy. A polymorphism affecting the gene encoding glycine amidinotransferase (GATM rs9806699 G > A) was previously associated with reduced risk for SIM. Our objective was to replicate the GATM association in a large, multicenter SIM case-control study. Mild and severe SIM cases and age- and gender-matched controls were enrolled. Participants were genotyped, and associations were tested (n = 715) using chi-square and logistic regression with consideration for SIM severity and exclusion of subjects with potentially confounding comedications. The minor allele (A) frequencies of GATM rs9806699 in the controls (n = 106), mild SIM (n = 324), and severe SIM (n = 285) cases were 0.26, 0.28, and 0.29, respectively (p = 0.447). The unadjusted odds ratio for the A allele for any SIM (mild or severe) was 1.14 (0.82-1.61; p = 0.437), which remained nonsignificant in all models. Our results do not replicate the association between GATM rs9806699 and SIM.

Antineuronal antibodies in idiopathic achalasia and gastro-oesophageal reflux disease.

BACKGROUND AND AIMS: The precise aetiology of achalasia is unknown although autoimmunity has been implicated and is supported by several studies. We screened sera from patients with achalasia or gastro-oesophageal reflux disease (GORD) to test for circulating antimyenteric neuronal antibodies. METHODS: Serum was obtained from 45 individuals with achalasia, 16 with GORD, and 22 normal controls. Serum was used in immunohistochemistry to label whole mount preparations of ileum and oesophagus of the guinea pig and mouse. Also, sections of superior cervical and dorsal root ganglia, and spinal cord were examined. RESULTS: Positive immunostaining of the myenteric plexus was detected in significantly more achalasia and GORD samples than control samples (achalasia, p<0.001; GORD, p<0.01), and immunoreactivity was significantly more intense with achalasia and GORD serum samples than controls (achalasia, p<0.01; GORD, p<0.05). There was no correlation between intensity of immunoreactivity and duration of achalasia symptoms. In most cases, achalasia and GORD sera stained all ileal submucosal and myenteric neurones, and oesophageal neurones. Immunostaining was not species specific; however, immunostaining was largely specific for enteric neurones. Western blot analysis failed to reveal specific myenteric neuronal proteins that were labelled by antibodies in achalasia or GORD serum. CONCLUSIONS: These data suggest that antineuronal antibodies are generated in response to tissue damage or some other secondary phenomenon in achalasia and GORD. We conclude that antineuronal antibodies found in the serum of patients with achalasia represent an epiphenomenon and not a causative factor.

Dansyl fluoride, a fluorescent inhibitor for the location of tumour cells in human tissues.

Dansyl fluoride (Dan-F), an active site directed fluorescent inhibitor of guanidinobenzoatase (GB), has been used for the location of tumour cells in frozen sections of human squamous cell carcinoma and colonic carcinoma tissues. The tumour cell surfaces having active GB bind Dan-F and fluoresce blue. The surrounding normal epithelial lung cell surfaces fail to bind Dan-F and hence lack fluorescence, whilst the normal colon cell surfaces have another isoenzymic form of GB, bind Dan-F and fluoresce blue. Kinetic studies have shown that Dan-F is an irreversible inhibitor of GB, and Dan-GB complexes are not dissociated with SDS and high salt concentration. However hydroxylamine (1 M) can dissociate Dan-GB complexes in the presence of 0.1% SDS, both on membrane-bound and in free solution. These studies suggest that Dan-F is a potent inhibitor of GB, and in very low concentration (3 x 10(-8) M) can be used as a novel fluorescent probe for the location of tumour cells in histological sections of human tissues.

Location of tumour cells in colon tissue by Texas red labelled pentosan polysulphate, an inhibitor of a cell surface protease.

Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon.

Interaction of tissue plasminogen activator inhibitor with cell surface guanidinobenzoatase and urokinase plasminogen activator.

This study employs fluorescent inhibitor molecules to detect both cell surface proteases and their receptor sites on colonic carcinoma cells. Present studies are concerned with the interactions of the tumour associated proteases, guanidinobenzoatase (GB) and plasminogen activators (PAs) with PAs inhibitor type 1 (PAI-1). The active enzymes on the cell surfaces in frozen sections of human colonic carcinoma tissue were located by staining with two active site directed fluorescent inhibitors, 9-aminoacridine (9-AA) and Rhodamine labelled PAI-1 (Rh-PAI-1), followed by fluorescence microscopy. Fibrin treated sections, which now lack GB but have receptor proteins for GB, fail to bind 9-AA and Rh-PAI-1. When these fibrin-treated sections were incubated with purified colonic carcinoma GB and u-PA, both enzymes were bound to the tumour cells in these sections and subsequent challenging with fluorescent probes for GB resulted in bright fluorescence under appropriate microscopic conditions. On the other hand when fibrin treated sections were incubated with t-PA, followed by challenging with Rh-PAI-1, no red fluorescence was observed. It is suggested that the GB and u-PA have similar specific binding sites which can recognise and bind to the receptors on tumour cells in fibrin-treated sections, but t-PA has no such binding site and fails to recognise the cell surface receptors for GB. These GB-receptors may have a possible role in the regulation of GB and u-PA activity during tumour cell invasion and metastasis.

Selectivity of the plasminogen activator inhibitor (PAI-1) for the iso enzyme of guanidinobenzoatase on the surface of colonic carcinoma cells.

The interaction of plasminogen activator-inhibitor (PAI-1) with a cell surface protease, guanidinobenzoatase (GB), has been studied in free solution and on the surface of colonic epithelial cells. It has been demonstrated that PAI-1 recognises and inhibits the iso enzymic form of GB associated with colonic carcinoma cells but fails to bind to the iso enzymic form of GB associated with normal donor colonic epithelial cells. This interaction is mediated by a lysyl binding site on the GB: complex formation prevents GB binding to fibrin fibrils which also involves lysyl binding sites.

Differential competitive inhibition of a cell surface protease on normal epithelial cells and carcinoma cells of the colon.

The cell surfaces of normal colonic epithelial cells and colonic carcinoma cells both possess a protease referred to as guanidinobenzoatase (GB). Previous studies have shown that these cells possess two distinct isoenzymic forms of GB which could be distinguished by their selective recognition of cytoplasmic protein inhibitors of GB. In the present study we have used competitive inhibitors of GB to demonstrate the differential inhibition of the GB on normal colonic epithelial cells whilst the GB on colonic carcinoma cell surfaces remains active. The enzymic status of GB on these cells has been determined by challenging the treated cells in frozen sections with a second fluorescent inhibitor, followed by fluorescence microscopic analysis.

Retinoic acid inhibition of a tumour protease immobilised on cell surfaces and in free solution.

Retinoids are inhibitors of tumour cell proliferation in culture and have been shown to suppress carcinogenesis and decrease the levels of proteases. The present study has demonstrated that retinoic acid is a potential non-competitive inhibitor of a protease (GB) immobilised on the surfaces of tumour cells in thin sections and free GB in solution. The enzymic status of GB on the cell surfaces in sections has been determined by challenging the retinoic acid-treated cells with a second fluorescent inhibitor (9-AA), followed by fluorescence microscopic analysis. The inhibition of cell surface GB by retinoic acid was demonstrated to be reversible. The activity of soluble GB has been measured by the MUGB assay in the presence and absence of retinoic acid. It is suggested that retinoic acid acts on GB by interacting with a binding site, different from the active site, and causes major conformational changes, resulting in enzyme inhibition. It is possible that the modulation of GB activity by retinoic acid may play a role in the control of cell migration and metastasis.