Cost-effectiveness analysis of atezolizumab plus bevacizumab compared with sorafenib as first-line treatment in advanced hepatocellular carcinoma in Singapore.

OBJECTIVES: This study aims to explore the cost-effectiveness of atezolizumab plus bevacizumab against sorafenib for first-line treatment of locally advanced or metastatic hepatocellular carcinoma (HCC) in Singapore. METHODS: A partitioned survival model was developed from a healthcare system perspective, with a 10-year lifetime horizon. Clinical inputs and utilities were obtained from the IMbrave150 trial. Healthcare resource use costs were obtained from published local sources; drug costs reflected the most recent public hospital selling prices. Outcomes included life years, quality-adjusted life years (QALYs) and incremental cost-effectiveness ratios (ICERs). Deterministic and probabilistic sensitivity analyses were performed to assess the model's robustness. RESULTS: Atezolizumab plus bevacizumab offered an additional 1.42 life years and 1.09 QALYs, with an additional cost of S$111,847; the ICER was S$102,988/QALY. The World Health Organization considers interventions with ICERs <1 gross domestic product (GDP)/capita to be highly cost-effective. At a willingness-to-pay (WTP) threshold of S$114,165/QALY (Singapore's 2022 GDP/capita), atezolizumab plus bevacizumab is cost-effective compared with sorafenib. The ICER was most sensitive to variations in utilities, but all parameter variations had no significant impact on the model outcomes. CONCLUSION: At a WTP threshold of Singapore's GDP/capita, atezolizumab plus bevacizumab is cost-effective compared with sorafenib.

Cost-effectiveness analysis of add-on pertuzumab to trastuzumab biosimilar and chemotherapy as neoadjuvant treatment for human epidermal growth receptor 2-positive early breast cancer patients in Singapore.

OBJECTIVES: The Asian PEONY trial showed that add-on pertuzumab to trastuzumab and chemotherapy significantly improved pathological complete response in the neoadjuvant treatment of patients with human epidermal growth factor receptor 2-positive (HER2+) early breast cancer (EBC). This study evaluated the cost-effectiveness of pertuzumab as an add-on therapy to trastuzumab and chemotherapy for neoadjuvant treatment of patients with HER2+ EBC in Singapore. METHODS: A six-state Markov model was developed from the Singapore healthcare system perspective, with a lifetime time horizon. Model outputs were: costs; life-years (LYs); quality-adjusted LYs (QALYs); incremental cost-effectiveness ratios (ICERs). Sensitivity/scenario analyses explored model uncertainties. RESULTS: The base case projected the addition of pertuzumab to be associated with improved outcomes by 0.277 LYs and 0.271 QALYs, increased costs by S$1,387, and an ICER of S$5,121/QALY. The ICER was most sensitive to the pCR rate, and the probabilistic sensitivity analysis showed that add-on pertuzumab had an 81.3% probability of being cost-effective at a willingness-to-pay threshold of S$45,000/QALY gained. CONCLUSIONS: This model demonstrated that the long-term clinical impact of early pertuzumab use, particularly the avoidance of metastatic disease and thus avoidance of higher costs and mortality rates, make neoadjuvant pertuzumab a cost-effective option in the management of patients with HER2+ breast cancer in Singapore.

Ultra-Short Antimicrobial Peptoids Show Propensity for Membrane Activity Against Multi-Drug Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) results in both morbidity and mortality on a global scale. With drug resistance on the increase, there is an urgent need to develop novel anti-mycobacterials. Thus, we assessed the anti-mycobacterial potency of three novel synthetic peptoids against drug-susceptible and multi-drug resistant (MDR) Mycobacterium tuberculosis in vitro using Minimum Inhibitory Concentration, killing efficacy and intracellular growth inhibition assays, and in vivo against mycobacteria infected BALB/c mice. In addition, we verified cell selectivity using mammalian cells to assess peptoid toxicity. The mechanism of action was determined using flow cytometric analysis, and microfluidic live-cell imaging with time-lapse microscopy and uptake of propidium iodide. Peptoid BM 2 demonstrated anti-mycobacterial activity against both drug sensitive and MDR M. tuberculosis together with an acceptable toxicity profile that showed selectivity between bacterial and mammalian membranes. The peptoid was able to efficiently kill mycobacteria both in vitro and intracellularly in murine RAW 264.7 macrophages, and significantly reduced bacterial load in the lungs of infected mice. Flow cytometric and time lapse fluorescence microscopy indicate mycobacterial membrane damage as the likely mechanism of action. These data demonstrate that peptoids are a novel class of antimicrobial which warrant further investigation and development as therapeutics against TB.

Galleria mellonella: An Infection Model for Screening Compounds Against the Mycobacterium tuberculosis Complex.

Drug screening models have a vital role in the development of novel antimycobacterial agents which are urgently needed to tackle drug-resistant tuberculosis (TB). We recently established the larvae of the insect Galleria mellonella (greater wax moth) as a novel infection model for the Mycobacterium tuberculosis complex. Here we demonstrate its use as a rapid and reproducible screen to evaluate antimycobacterial drug efficacy using larvae infected with bioluminescent Mycobacterium bovis BCG lux. Treatment improved larval survival outcome and, with the exception of pyrazinamide, was associated with a significant reduction in in vivo mycobacterial bioluminescence over a 96 h period compared to the untreated controls. Isoniazid and rifampicin displayed the greatest in vivo efficacy and survival outcome. Thus G. mellonella, infected with bioluminescent mycobacteria, can rapidly determine in vivo drug efficacy, and has the potential to significantly reduce and/or replace the number of animals used in TB research.

Facile and efficient encapsulation of antimicrobial peptides via crosslinked DNA nanostructures and their application in wound therapy.

There is growing interest in the development of nucleic acid nanostructures as smart functional materials for applications in drug delivery. Inspired by the diverse physical interactions that exist in nature, crosslinked DNA nanostructures can serve as attractive affinity binding networks that interact with therapeutic cargos or living cells. Herein we report a strategy that addresses the challenges of topical oligopeptide therapy by exploiting high binding affinity between polyanionic DNA nanostructures and cationic antimicrobial peptides (AMPs) to fabricate hydrogels that release a model antimicrobial L12 peptide in response to pathogenic S. aureus infections. We further demonstrated controlled peptide release profiles via the DNA hydrogels that were biocompatible and delivered superior antimicrobial activity against nuclease-releasing susceptible and methicillin-resistant S. aureus infections. Single application of the L12-loaded DNA hydrogels on porcine explant S. aureus infections revealed potent efficacy after 24h. As a result of the capacity of the crosslinked DNA nanostructures to elicit a strong anti-inflammatory response, in vivo treatment of mice excision wounds translated into faster healing rates. Overall, the crosslinked DNA nanostructures reported in this study offer significant advantage as functional wound dressings and their future adaptation holds equally great promise for the delivery of cationic antimicrobials.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world's population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Antimicrobial and Anti-Biofilm Activities of Surface Engineered Polycationic Albumin Nanoparticles with Reduced Hemolytic Activity.

Protein-based polymeric polyelectrolytes are emerging as alternative synthetic nanoparticles owing to their biodegradability and biocompatibility. However, potential in vivo toxicity remains a significant challenge. Herein an array of protein polyelectrolytes generated from cationic human serum albumin (cHSA) and polyethylene glycol (PEG) are synthesized via synthetic customization as antimicrobials for the treatment of systemic infections. By varying PEG molecular weight and chain length, in vitro hemolytic activity can be fine-tuned without significantly affecting antimicrobial potency. The optimal hybrid material, PEG (2000)18 -cHSA, with potent antimicrobial character, low hemolytic activity, and in vitro biofilm disruptive properties is identified. Surface plasmon resonance (SPR) evaluation demonstrates significantly higher binding activity of the protein nanoparticles to bacteria cell wall components and microfluidic live-cell imaging indicates that the nanoparticles act through a membranolytic mechanism. Given their low susceptibility to drug resistance and potent activity against resistant bacteria strains, these findings establish the PEGylated albumin nanoparticles as a potent weaponry against drug resistance and biofilm-related infection.

Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex.

Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the Mycobacterium tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection.

Disruption of drug-resistant biofilms using de novo designed short α-helical antimicrobial peptides with idealized facial amphiphilicity.

The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X1Y1Y2X2)n, where X1 and X2 are hydrophobic residues (Leu or Ile or Trp), Y1 and Y2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL)3, promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL)3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2h of treatment. The peptides (LKKL)3 and (WKKW)3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections. STATEMENT OF SIGNIFICANCE: Antimicrobial peptides (AMPs) are increasingly explored as therapeutics for drug-resistant and biofilm-related infections to help expand the size and quality of the current antibiotic pipeline in the face of mounting antimicrobial resistance. Here, synthetic peptides rationally designed based upon principles governing the folding of natural α-helical AMPs, comprising the backbone sequence (X1Y1Y2X2)n, and which assemble into α-helical structures with idealized facial amphiphilicity, is presented. These multifunctional peptide amphiphiles demonstrate high bacterial selectivity, promote the disruption of pre-formed drug-resistant biofilms, and effectively neutralize endotoxins at low micromolar concentrations. Overall, the design strategies presented here could provide a useful tool for developing therapeutic peptides with broad-ranging clinical applications from the treatment and prevention of drug-resistant biofilms to the neutralization of bacterial endotoxins.

Biodegradable functional polycarbonate micelles for controlled release of amphotericin B.

Amphotericin B (AmB), a poorly soluble and toxic antifungal drug, was encapsulated into polymeric micelles self-assembled from phenylboronic acid-functionalized polycarbonate/PEG (PEG-PBC) and urea-functionalized polycarbonate/PEG (PEG-PUC) diblock copolymers via hydrogen-bonding, boronate ester bond, and/or ionic interactions between the boronic acid group in the micellar core and amine group in AmB. Three micellar formulations were prepared: AmB/B micelles using PEG-PBC, AmB/U micelles using PEG-PUC and AmB/B+U mixed micelles using 1:1molar ratio of PEG-PBC and PEG-PUC. The average particle sizes of the micelles were in the range of 54.4-84.8nm with narrow size distribution and zeta potentials close to neutral. UV-Vis absorption analysis indicated that AmB/B micelles significantly reduced AmB aggregation status due to the interactions between AmB and the micellar core, while Fungizone® and AmB/U micelles had no effect. AmB/B+U mixed micelles exerted an intermediate effect. Both AmB/B micelles and AmB/B+U mixed micelles showed sustained drug release, with 48.6±2.1% and 59.2±1.8% AmB released respectively after 24hunder sink conditions, while AmB/U micelles displayed a burst release profile. All AmB-loaded micelles showed comparable antifungal activity to free AmB or Fungizone®, while AmB/B micelles and AmB/B+U mixed micelles were much less hemolytic than other formulations. Histological examination showed that AmB/B and AmB/B+U micelles led to a significantly lower number of apoptotic cells in the kidneys compared to Fungizone®, suggesting reduced nephrotoxicity of the micellar formulations in vivo. These phenylboronic acid-functionalized polymeric micelle systems are promising drug carriers for AmB to reduce non-specific toxicities without compromise in antifungal activity. STATEMENT OF SIGNIFICANCE: There is a pressing need for a novel and cost-effective delivery system to reduce the toxicity induced by the antifungal agent, amphotericin B (AmB). In this study, phenylboronic acid-functionalized polycarbonate/PEG diblock copolymers were used to fabricate micelles for improved AmB-micelle interaction via the manipulation of hydrogen-bonding, boronate ester bond, ionic and hydrophobic interactions. Compared to free AmB and Fungizone®, the resultant micellar systems displayed improved stability while reducing non-specific toxicities without a compromise in antifungal activity. These findings demonstrate the potential of biodegradable functional polycarbonate micellar systems as promising carriers of AmB for the treatment of systemic fungal infections.

Stability and efficacy of synthetic cationic antimicrobial peptides nebulized using high frequency acoustic waves.

Surface acoustic wave (SAW), a nanometer amplitude electroelastic wave generated and propagated on low-loss piezoelectric substrates (such as LiNbO3), is an extremely efficient solid-fluid energy transfer mechanism. The present study explores the use of SAW nebulization as a solution for effective pulmonary peptide delivery. In vitro deposition characteristics of the nebulized peptides were determined using a Next Generation Cascade Impactor. 70% of the peptide-laden aerosols generated were within a size distribution favorable for deep lung distribution. The integrity of the nebulized peptides was found to be retained, as shown via mass spectrometry. The anti-mycobacterial activity of the nebulized peptides was found to be uncompromised compared with their non-nebulized counterparts, as demonstrated by the minimum inhibition concentration and the colony forming inhibition activity. The peptide concentration and volume recoveries for the SAW nebulizer were significantly higher than 90% and found to be insensitive to variation in the peptide sequences. These results demonstrate the potential of the SAW nebulization platform as an effective delivery system of therapeutic peptides through the respiratory tract to the deep lung.

Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability.

OBJECTIVES: The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities. METHODS: Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy. RESULTS: Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all d-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified l-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm. CONCLUSIONS: Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.

Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis: Discerning the role of hydrophobicity and helicity.

Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant Mycobacterium tuberculosis. In this study, the effect of key physicochemical parameters including hydrophobicity and helicity of α-helical peptides on anti-mycobacterial activity and synergism with rifampicin was investigated. The most hydrophobic analogue, W(LLKK)2W, displayed low selectivity against mycobacteria while peptides with intermediate hydrophobicity were shown to be equally active, yet significantly less toxic. Furthermore, proline substitution impeded the formation of stable amphipathic structures, rendering P(LLKK)2P as one of the least active analogues. Terminal capping with isoleucine was found to promote α-helical folding and the resultant peptide demonstrated the highest selectivity and minimal cytotoxicity against mammalian macrophages. Flow cytometric analysis revealed that enhancements in hydrophobicity and α-helicity increased the rate and extent of peptide-mediated membrane permeabilization. This finding corroborated the hypothesis that synergism between the peptides and rifampicin was likely mediated via peptide-induced pore formation. The rapid, concentration-dependent membrane depolarization, leakage of intracellular ATP and calcein release from PE/PG LUVs supported the membrane-lytic mechanism of action of the peptides. Together, these findings suggest that hydrophobicity and α-helicity significantly impact anti-mycobacterial activity and optimization of both parameters is necessary to develop synthetic analogues with superior selectivity indices and enhanced synergistic potential with conventional antibiotics. STATEMENT OF SIGNIFICANCE: There is an urgent clinical need for the discovery of new antimicrobials, effective not just for drug susceptible, but also rapidly emerging drug-resistant TB. Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant M. tuberculosis. In this study, we evaluated a series of synthetic α-helical (LLKK)2 peptides over a range of hydrophobicities for their activity against mycobacteria and provide the first report on the modulating effect of hydrophobicity and α-helicity on the antimicrobial mechanisms of synthetic AMPs and their synergism with first-line antibiotics. These findings demonstrate the applicability of strategies employed here for the rational design of AMPs with the aim of improving cell selectivity and synergistic interactions when co-administered with first-line antibiotics in the fight against drug-resistant tuberculosis.