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Multi-Methodological Quantitative Taste Assessment of Anti-Tuberculosis Drugs to Support the Development of Palatable Paediatric Dosage Forms
No full textThe unpalatability of antituberculosis drugs is often cited as a major cause of non-adherence
in children, yet limited quantitative taste assessment data are available. The aim of this research was to quantify the bitterness of isoniazid, rifampicin, pyrazinamide, and ethambutol dihydrochloride
using two in vivo (a human taste panel and a rat brief-access taste aversion (BATA) model) and one in vitro (sensor) method. The response of the Insent TS-5000Z electronic tongue was compared to the in vivo drug concentration found to elicit and suppress half the maximum taste response (EC50 in human and IC50 in rats). Using dose-relevant concentrations, an overarching rank order of bitterness was derived (rifampicin > ethambutol > pyrazinamid~isoniazid). In vitro, only ethambutol exhibited a linear response for all sensors/concentrations. Based on the EC50/IC50 generated, a ‘taste index’ was proposed to allow for anticipation of the likelihood of taste issues in practice, taking in account the saturability in the saliva and therapeutic doses; ethambutol and isoniazid were found to be the worst tasting using this measure. The study presents the first quantitative taste analysis of these life-saving drugs and has allowed for a comparison of three methods of obtaining such data. Such information allows the operator to identify and prioritise the drugs requiring taste masking to produce palatable formulations
TNF-α antagonists differentially induce TGF-β1-dependent resuscitation of dormant-like Mycobacterium tuberculosis
No full textTNF-α- as well as non-TNF-α-targeting biologics are prescribed to treat a variety of immune-mediated inflammatory disorders. The well-documented risk of tuberculosis progression associated with anti-TNF-α treatment highlighted the central role of TNF-α for the maintenance of protective immunity, although the rate of tuberculosis detected among patients varies with the nature of the drug. Using a human, in-vitro granuloma model, we reproduce the increased reactivation rate of tuberculosis following exposure to Adalimumab compared to Etanercept, two TNF-α-neutralizing biologics. We show that Adalimumab, because of its bivalence, specifically induces TGF-β1-dependent Mycobacterium tuberculosis (Mtb) resuscitation which can be prevented by concomitant TGF-β1 neutralization. Moreover, our data suggest an additional role of lymphotoxin-α–neutralized by Etanercept but not Adalimumab–in the control of latent tuberculosis infection. Furthermore, we show that, while Secukinumab, an anti-IL-17A antibody, does not revert Mtb dormancy, the anti-IL-12-p40 antibody Ustekinumab and the recombinant IL-1RA Anakinra promote Mtb resuscitation, in line with the importance of these pathways in tuberculosis immunity
Lerisetron Analogues with Antimalarial properties - Synthesis, Structure-Activity Relationship (SAR) studies and Biological Assessment
No full textA phenotypic whole cell high throughput screen against asexual blood and liver stages of the malaria parasite identified a benzimidazole chemical series. Among the hits were the antiemetic benzimidazole drug Lerisetron 1 (IC50 NF54 = 0.81 µM) and its methyl substituted analogue 2 (IC50 NF54 = 0.098 µM). A medicinal chemistry hit to lead effort led to the identification of chloro substituted analogue 3 with high potency against the drug sensitive NF54 (IC50 NF54 = 0.062 µM) and multidrug-resistant K1 (IC50 K1= 0.054 µM) strains of the human malaria parasite Plasmodium falciparum. Cardiotoxicity risks as expressed in strong inhibition of the hERG potassium channel was identified as major liability to address. This led to the synthesis and biological assessment of around sixty (60) analogues from which several compounds with more potent anti-plasmodium activity, relative to the lead compound 3, were identified
Promiscuity analysis of a kinase panel screen with designated p38 alpha inhibitors
No full textProtein phosphorylation by kinases is of critical importance for the regulation of many cellular functions. When kinases are deregulated numerous biological processes are affected, which may cause a variety of diseases. Therefore, kinase inhibition plays an important role for therapeutic intervention. A number of kinase inhibitors have been approved as drugs, initially in oncology where promiscuous (multi-kinase) inhibitors were most efficacious. Exploring kinase inhibitor selectivity and promiscuity for therapy is among the most challenging aspects of kinase drug discovery. Herein, we thoroughly analyze a kinase profiling experiment in which 637 designated inhibitors of p38α MAP kinase (p38α) were tested against a panel of 60 kinases distributed across the human kinome. In this experiment, only 19% of the inhibitors were found to be promiscuous when the median p38α inhibition level was applied as an activity threshold. Promiscuous inhibitors had a median value of two targets per compound, and many of these inhibitors were only active against the p38α and closely related JNK3 enzymes. Promiscuity cliffs were identified and analyzed in a network representation revealing structural modifications that were implicated in triggering compound promiscuity. Taken together, the findings revealed a high degree of selectivity of designated p38α directed inhibitors although they target the ATP binding site that is largely conserved across the human kinome
A novel luminescence-based high-throughput approach for cellular resolution of protein ubiquitination using tandem ubiquitin binding entities (TUBEs).
No full textProtein turnover is highly regulated by the post-translational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. Development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is essential for drug discovery in the UPS. Conventional approaches for studying ubiquitination either require high amounts of starting protein or rely on exogenous or modified ubiquitin moieties and thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay which combines the NanoBiT luminescence-based technology with tandem ubiquitin-binding entities (TUBE) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied the compound-induced ubiquitination of G To S Phase Transition 1 (GSPT1) protein. Using this new assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a novel cell based approach for assaying substrate ubiquitination in living cells that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying ubiquitination of target proteins with varying functions
Simple synthesis of amides via their acid chloride in aqueous TPGS-750-M
No full textThe technology of surfactant chemistry is employed for amide bond construction via the reaction of acyl chlorides
with amines in 2 wt % TPGS-750-M aqueous solution. Specifically, this highly efficient method enables a chromatography-free scalable process and recycling of the TPGS-750-M solution
LGR5 controls extracellular matrix production by stem cells in the developing intestine
No full textThe Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs
Physiologically Based Pharmacokinetic Modeling of Oral Absorption, pH, and Food Effect in Healthy Volunteers to Drive Alpelisib Formulation Selection
No full textA physiologically based pharmacokinetic (PBPK) human model for alpelisib, an oral α-specific class I phosphatidylinositol-3-kinase (PI3K) inhibitor, was established to simulate oral absorption and plasma pharmacokinetics of healthy subjects to allow model-informed drug development. The GastroPlus™ model consisted of an advanced absorption gut model, which was linked to a 2-compartmental model. Systemic clearance and volume of distribution were estimated using population pharmacokinetics (popPK). Various food effect and pH-mediated absorption drug–drug interaction (DDI) scenarios were modeled. In fasted healthy subjects, simulated absorption was lower (ca. 70% for a 300-mg dose) due to pH and bile acid concentration-dependent solubility. Ranitidine showed a significant pH-mediated DDI effect only in the fasted but not fed state. The PBPK model identified that more drug is absorbed in the fed state, and alpelisib intestinal permeability is rate limiting to systemic exposure. Simulations for healthy subject showed a positive food effect with ca. 2-fold increase in plasma Cmax and 1.5-fold increase in AUC0-inf with a meal compared with fasted conditions. The PBPK model was verified using clinical food effect data with pivotal clinical formulation (PCF) and then applied to predict the performance of a commercial formulation (CF) in healthy volunteers. The model successfully predicted the outcome of a clinical bioequivalence study for PCF and CF with included in vitro dissolution data, both fasted and fed state. Estimated predictive errors (based on plasma Cmax, AUC0-t) were equal or below 30%. The alpelisib model for healthy subjects enables future bioequivalence formulation assessments, in fasted, fed, or altered pH conditions. [Figure not available: see fulltext.
A Cyclic Phosphoramidate Prodrug of 2'-deoxy-2'-fluoro-2'-C-methylguanosine for the Treatment of Dengue Infection
No full textMonophosphate prodrug analogs of 2'-deoxy-2'-fluoro-2'-C-methylguanosine have been reported as potent inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase. These prodrugs also display potent anti-dengue activities in cellular assays although their prodrug moieties were designed to produce high levels of triphosphate in the liver. Since peripheral blood mononuclear cells (PBMCs) are one of the major targets of dengue virus, different prodrug moieties were designed to effectively deliver 2'-deoxy-2'-fluoro-2'-C-methylguanosine monophosphate prodrugs and their corresponding triphosphates into PBMCs after oral administration. We identified a cyclic phosphoramidate prodrug 17 demonstrating a well-balanced anti-dengue cellular activity and in vitro stability profiles. We further determined the PBMCs concentration of active triphosphate needed to inhibit 50% virus replication (TP50). Compound 17 was assessed in AG129 mouse model and demonstrated 1.6- and 2.2-log viremia reduction at 100 and 300 mg/kg BID, respectively. At 100 mg/kg BID, the terminal triphosphate concentration in PBMCs reached above TP50, demonstrating TP50 as the target exposure for efficacy. In dogs, oral administration of 17 resulted in high PBMCs triphosphate level, exceeding TP50 at 10 mg/kg. Unfortunately, two-week dog toxicity studies at 30, 100, and 300 mg/kg/day showed that No Observed Adverse Effect Level (NOAEL) could not be achieved due to pulmonary inflammation and hemorrhage. The preclinical safety results suspended further development of 17 Nevertheless, present work has proven the concept that an efficacious monophosphate nucleoside prodrug could be developed for the potential treatment of dengue infection
Physiologically Based Pharmacokinetic Modeling of Oral Absorption, pH, and Food Effect in Healthy Volunteers to Drive Alpelisib Formulation Selection
No full textA physiologically based pharmacokinetic (PBPK) human model for alpelisib, an oral α-specific class I phosphatidylinositol-3-kinase (PI3K) inhibitor, was established to simulate oral absorption and plasma pharmacokinetics of healthy subjects to allow model-informed drug development. The GastroPlus™ model consisted of an advanced absorption gut model, which was linked to a 2-compartmental model. Systemic clearance and volume of distribution were estimated using population pharmacokinetics (popPK). Various food effect and pH-mediated absorption drug–drug interaction (DDI) scenarios were modeled. In fasted healthy subjects, simulated absorption was lower (ca. 70% for a 300-mg dose) due to pH and bile acid concentration-dependent solubility. Ranitidine showed a significant pH-mediated DDI effect only in the fasted but not fed state. The PBPK model identified that more drug is absorbed in the fed state, and alpelisib intestinal permeability is rate limiting to systemic exposure. Simulations for healthy subject showed a positive food effect with ca. 2-fold increase in plasma Cmax and 1.5-fold increase in AUC0-inf with a meal compared with fasted conditions. The PBPK model was verified using clinical food effect data with pivotal clinical formulation (PCF) and then applied to predict the performance of a commercial formulation (CF) in healthy volunteers. The model successfully predicted the outcome of a clinical bioequivalence study for PCF and CF with included in vitro dissolution data, both fasted and fed state. Estimated predictive errors (based on plasma Cmax, AUC0-t) were equal or below 30%. The alpelisib model for healthy subjects enables future bioequivalence formulation assessments, in fasted, fed, or altered pH conditions. [Figure not available: see fulltext.