In Situ Transformable Supramolecular Nanomedicine Targeted Activating Hippo Pathway for Triple-Negative Breast Cancer Growth and Metastasis Inhibition.

As it is closely associated with tumor proliferation, metastasis, and the immunosuppressive microenvironment, the dysfunctional Hippo pathway has become an extremely attractive target for treating multiple cancers. However, to date, the corresponding chemotherapeutic nanomedicines have not been developed. Herein, a supramolecular self-delivery nanomedicine with in situ transforming capacity was tailor-constructed for Hippo-pathway restoration, and its inhibitory effect against tumor growth and metastasis was investigated in a highly aggressive triple-negative breast cancer (TNBC) model. Stimulated by overexpressed glutathione (GSH) and esterase in cancer cells, the self-assembled nanomedicine transformed from inactive nanospheres to active nanofibers conjugating tyrosvaline and spatiotemporally synchronously released the covalently linked flufenamic acid in situ, together activating the maladjusted Hippo pathway by simultaneously acting on different targets upstream and downstream. The transcriptional expression of Yes-associated protein (YAP) and related growth-promoted genes were significantly reduced, finally significantly repressing the proliferation and metastasis of cancer cells. Additionally, the Hippo-pathway restoration showed an excellent radiosensitization effect, making the targeted therapy combined with radiotherapy display a prominent synergistic in vivo anticancer effect against TNBC. This work reports a specifically designed smart nanomedicine to restore the function of the Hippo pathway and sensitize radiotherapy, providing an attractive paradigm for targeted drug delivery and cancer combination therapy.

[Angioedema and anaphylaxis, a family story]. / Angiœdème et anaphylaxie, une histoire de famille.

Non-allergic angioedema has a worrying morbidity. Clinical examination is central, as C1-esterase inhibitor deficiency will not be documented in the acute phase. In the case of anaphylaxis that does not respond to adrenaline, an early diagnosis can optimise referral of the patient to a reference healthcare establishment for a specific therapeutic protocol (icatibant, C1 inhibitor) recently updated by recommendations.

Esterase-responsive and size-optimized prodrug nanoparticles for effective intracranial drug delivery and glioblastoma treatment.

Glioblastoma multiforme (GBM) is the intracranial malignancy with the highest rates of morbidity and mortality. Chemotherapy is often ineffective against GBM due to the presence of the blood-brain barrier (BBB); however, the application of nanotechnology is expected to overcome this limitation. Poly(lactic-co-glycolic acid) (PLGA) is a degradable and nontoxic functional polymer with good biocompatibility that is widely used in the pharmaceutical industry. Previous studies have shown that the ability of PLGA nanoparticles (NPs) to penetrate the BBB is largely determined by their size; however, determination of the optimal PLGA NP size requires further research. Here, we report a tandutinib-based prodrug (proTan), which responds to the GBM microenvironment, that was combined with NPs to overcome the BBB. AMD3100-PLGA NPs loaded with proTan inhibited tumor growth and effectively prolonged the survival of tumor-bearing mice.

Prophylactic use of an anti-activated factor XII monoclonal antibody, garadacimab, for patients with C1-esterase inhibitor-deficient hereditary angioedema: a randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Hereditary angioedema is associated with dysregulation of the kallikrein-kinin system. Factor XII (FXII) is a key initiator of the kallikrein-kinin system, which produces bradykinin, a central mediator of angioedema. Garadacimab (CSL Behring) is a first-in-class, fully human, immunoglobulin G4 monoclonal antibody targeting activated FXII, intended to prevent attacks in patients with C1-esterase inhibitor-deficient hereditary angioedema (HAE-C1-INH). We aimed to investigate garadacimab as a treatment every 4 weeks for patients with HAE-C1-INH. METHODS: In this double-blind, placebo-controlled, phase 2 study, patients with HAE-C1-INH were recruited from 12 research centres in Canada, Germany, Israel, and the USA. Eligible patients were aged 18-65 years and must have had at least four attacks of any severity over a consecutive 2-month period during the 3 months before screening or initiation of previous hereditary angioedema prophylaxis. After a run-in period of 4-8 weeks, patients were randomly assigned (1:1:1:1), using an interactive response technology via block randomisation (block sizes of 1-4), to either placebo or 75 mg, 200 mg, or 600 mg garadacimab. Patients were given an initial intravenous loading dose, and then, on day 6 and every 4 weeks for 12 weeks, they were given a subcutaneous dose of their allocated treatment. The primary endpoint was the number of monthly attacks in the intention-to-treat population (defined as all patients who underwent screening, provided consent, and were assigned to treatment) during the 12-week subcutaneous administration period assessed in the 200 mg and 600 mg garadacimab groups versus placebo. Safety was assessed in all patients who received at least one dose or partial dose of study treatment. This study is registered with ClinicalTrials.gov, NCT03712228. FINDINGS: Between Oct 29, 2018, and Aug 28, 2019, 54 patients were screened, of whom 32 were randomly assigned to either placebo (n=8) or 75 mg (n=9), 200 mg (n=8), or 600 mg (n=7) garadacimab. The median age was 39·5 years (28·0-52·5) and 18 (56%) of 32 patients were female and 14 (34%) were male. The median number of monthly attacks during the 12-week subcutaneous treatment period was 4·6 (IQR 3·1-5·0) with placebo, 0·0 (0·0-0·4) with 75 mg garadacimab, 0·0 (0·0-0·0) with 200 mg garadacimab, and 0·3 (0·0-0·7) with 600 mg garadacimab. Compared with placebo, the rate of attacks was significantly reduced with garadacimab at 200 mg (reduced by 100% [95% CI 98-101]; p=0·0002) and 600 mg (reduced by 93% [54-110]; p=0·0003). No serious adverse events, deaths, or adverse events of special interest (anaphylaxis, thromboembolic events, and bleeding events) were observed. INTERPRETATION: Garadacimab 200 mg and 600 mg every 4 weeks significantly reduced the number of monthly attacks versus placebo and was well tolerated during the study. Garadacimab is an efficacious, subcutaneous prophylaxis in patients with HAE-C1-INH and warrants phase 3 evaluation. FUNDING: CSL Behring.

Molecular dynamics and structure-based virtual screening and identification of natural compounds as Wnt signaling modulators: possible therapeutics for Alzheimer's disease.

Wnt signaling pathway is an evolutionarily conserved pathway responsible for neurogenesis, axon outgrowth, neuronal polarity, synapse formation, and maintenance. Downregulation of Wnt signaling has been found in patients with Alzheimer's disease (AD). Several experimental approaches to activate Wnt signaling pathway have proven to be beneficial in alleviating AD, which is one of the new therapeutic approaches for AD. The current study focuses on the computational structure-based virtual screening followed by the identification of potential phytomolecules targeting different markers of Wnt signaling like WIF1, DKK1, LRP6, GSK-3ß, and acetylcholine esterase. Initially, screening of 1924 compounds from the plant-based library of Zinc database was done for the selected five proteins using docking approach followed by MM-GBSA calculations. The top five hit molecules were identified for each protein. Based on docking score, and binding interactions, the top two hit molecules for each protein were selected as promising molecules for the molecular dynamic (MD) simulation study with the five proteins. Therefore, from this in silico based study, we report that Mangiferin could be a potential molecule targeting Wnt signaling pathway modulating the LRP6 activity, Baicalin for AChE activity, Chebulic acid for DKK1, ZINC103539689 for WIF1, and Morin for GSk-3ß protein. However, further validation of the activity is warranted based on in vivo and in vitro experiments for better understanding and strong claim. This study provides an in silico approach for the identification of modulators of the Wnt signaling pathway as a new therapeutic approach for AD.

Nanoparticles for Cancer Diagnosis, Radionuclide Therapy and Theranostics.

Nanoparticles have unique properties that can be exploited for cancer diagnosis and therapy. Intravenously injected nanoparticles accumulate predominantly in organs of the mononuclear phagocytic system, in addition to localizing in tumors and at sites of inflammation and infection. Accumulation in the liver and spleen lowers nanoparticles' ability to target pathological sites and compromises their use for radionuclide therapy. As described by Lee et al. in this issue of ACS Nano, radionuclide retention in liver and spleen can be greatly reduced by using liposomes that are surface-modified with esterase-cleavable radionuclide anchors. Because esterase activity is high in healthy tissues and low in tumors, the authors found that liposome-associated radioactivity rapidly cleared from the body and remained high only in tumors. The resulting images had high contrast-to-background ratios and remarkable tumor delineation. In this Perspective, we discuss these advances from early detection, cancer diagnosis, radionuclide therapy, and theranostics points of view. We outline the current clinical landscape of radionuclide targeting, imaging and therapy, and reflect on the roles that nanoparticles can play in these applications. We highlight the potential of nanoparticles that are responsive to endogenous stimuli for intraoperative imaging and, particularly, for individualized and improved radionuclide treatment. Taking these advances into account, future studies exploring the robustness and the clinical feasibility of nanomedicine-based radiotheranostic probes are eagerly awaited.

Paraoxonase and coronary heart disease.

The antioxidant activity of HDL is largely due to the paraoxonase (PON1) located on it. Experiments with transgenic PON1 knock-out mice indicate the potential for PON1 to protect against atherogenesis. This effect of HDL in decreasing LDL lipid peroxidation is maintained for longer than that of antioxidant vitamins and could thus be more protective. Several important advances in the field of PON research have occurred recently, not least the discovery that two other members of the PON gene family PON2 and PON3 may also have important antioxidant properties. Significant advances have been made in understanding the basic biochemical function of PON1 and the discovery of possible modulators of its activity. Decreased coronary heart disease (CHD) risk associated with polymorphisms of PON1 which are most active in lipid peroxide hydrolysis revealed by meta-analysis is likely to be an underestimate of the true contribution of PON1 to CHD because these polymorphisms explain only a small component of the variation in PON1 activity. However, it is a very important observation because genetic influences are not likely to be confounded by other factors linked with both CHD and diminished PON1 activity. PON1 is extensively researched and strategies will hopefully emerge to increase its activity and provide a more satisfactory test of the antioxidant hypothesis of atherosclerosis than antioxidant vitamins have done.

Success of pyridostigmine, physostigmine, eptastigmine and phosphotriesterase treatments in acute sarin intoxication.

The acute toxicity of organophosphorus (OP) compounds in mammals is due to their irreversible inhibition of acetylcholinesterase (AChE) in the nervous system, which leads to increased synaptic acetylcholine levels. The protective actions of intravenously (i.v.) administered pyridostigmine, physostigmine, eptastigmine, and an organophosphate hydrolase, phosphotriesterase, in acute sarin intoxication were studied in mice. The acute intragastric (i.g.) toxicity (LD50) of sarin with and without the pretreatments was tested by the up-and-down method. The mice received pyridostigmine (0.06 mg/kg body weight), physostigmine (0.09 mg/kg body weight), the physostigmine derivative eptastigmine (0.90 mg/kg body weight) or phosphotriesterase (104 U/g, 10.7 microg/g body weight) 10 min prior to the i.g. administration of sarin. Physostigmine was also administered with phosphotriesterase. Phosphotriesterase was the most effective antidote in sarin intoxication. The LD50 value for sarin increased 3.4-fold in mice receiving phosphotriesterase. Physostigmine was the most effective carbamate in sarin exposure. The protective ratios of physostigmine and pyridostigmine were 1.5- and 1.2-1.3-fold, respectively. Eptastigmine did not give any protection against sarin toxicity. Both the phosphotriesterase and physostigmine treatments protected the brain AChE activities measured 24 h after sarin exposure. In phosphotriesterase and physostigmine-treated mice, a 4- and 2-fold higher sarin dose, respectively, was needed to cause a 50% inhibition of brain AChE activity. Moreover, the combination of phosphotriesterase-physostigmine increased the LD50 value for sarin 4.3-fold. The animals pretreated with phosphotriesterase-ephysostigmine tolerated four times the lethal dose in control animals, furthermore their survival time was 2-3 h in comparison to 20 min in controls. In conclusion, phosphotriesterase and physostigmine were the most effective treatments against sarin intoxication. However, eptastigmine did not provide any protection against sarin toxicity.

Eptastigmine-phosphotriesterase combination in DFP intoxication.

A novel therapy against organophosphate exposure, the combination of a carbamate eptastigmine and an organophosphate hydrolase (phosphotriesterase) was studied in mice against diisopropylfluorophosphate (DFP) (1.75 mg/kg) exposure. Mice received eptastigmine (0.9 mg/kg; iv) 10 min prior to the ip injection of DFP. Phosphotriesterase (83 U/g body weight) was injected iv 10 min after DFP. Eptastigmine (1.5 mg/kg; iv) inhibited the acetylcholinesterase activities in brain and erythrocytes for a longer time than physostigmine. Eptastigmine caused only minor changes in the behavior and activity of the animals, whereas physostigmine clearly reduced their activity for about 30 min. The eptastigmine pretreatment clearly supplemented the protective effect of phosphotriesterase against DFP: the plasma butyrylcholinesterase activity was doubled and the activity recovered faster than in animals treated with phosphotriesterase alone. In lung, butyrylcholinesterase activity was initially lower after eptastigmine-phosphotriesterase than phosphotriesterase treatment alone. However, the activity returned 24 hr later to normal in eptastigmine-phosphotriesterase-treated groups. With phosphotriesterase only, it recovered only to 75% of the control level. Presumably eptastigmine, by preventing the binding of DFP to cholinesterases, caused an elevation of free DFP levels in body fluids and promoted phosphotriesterase hydrolysis of DFP.

Evaluation of digestive proteinases from the Antarctic krill Euphasia superba as potential chemonucleolytic agents. In vitro and in vivo studies.

Chemonucleolysis is a therapeutic procedure whereby a degradative enzyme is injected intradiscally to reduce disc height/width by depolymerisation of extracellular matrix components. This process is considered to diminish disc pressure on inflamed nerve roots, resulting in the alleviation of sciatic pain. In the present study two krill (Euphasia superba) enzyme preparations, a proteinase and an esterase preparation, were evaluated for their potential as chemonucleolytic agents. Initially, their ability to degrade several protein (azocoll, casein, proteoglycans, PGs) and peptide (CBZ-arg-4-nitroanilide, CBZ-lys-thiobenzyl ester) substrates was assessed in vitro. The krill proteinase preparation rapidly converted azocoll, casein and PGs to small peptides. Furthermore, when this degradative enzyme preparation was evaluated in vivo, a relatively low intradiscal dose (0.54 mg/disc) was found to reduce intervertebral disc widths in beagles to 48% +/- 10.5% (mean +/- SEM) of their pre-injection values within 2 weeks of administration. Moreover, the discs injected with this proteinase had reconstituted up to 80% +/- 9% (mean +/- SEM) of their pre-injection widths at the termination of the experiment (32 weeks). These data suggest that the krill protease preparation has potential as a chemonucleolytic agent which would allow disc matrix reconstitution. Conversely, the krill esterase preparation also degraded PGs, but into relatively large fragments. This limited digestion of PGs indicates that the krill esterase would be a less effective chemonucleolytic agent than the corresponding proteinase.

Encapsulation of phosphotriesterase within murine erythrocytes.

A new conceptual approach was employed to antagonize organophosphorus intoxication by using resealed carrier erythrocytes containing a recombinant phosphotriesterase. This enzyme has been reported to hydrolyze many organophosphorus compounds, including paraoxon, a potent cholinesterase inhibitor. Paraoxon is rapidly hydrolyzed by this enzyme to p-nitrophenol and diethylphosphate. Incorporation of phosphotriesterase within resealed murine erythrocytes was accomplished by hypotonic dialysis. The properties of this enzyme within these resealed erythrocytes were investigated. Addition of paraoxon to reaction mixtures containing these resealed erythrocytes loaded with phosphotriesterase resulted in the rapid hydrolysis of paraoxon. Hydrolysis of paraoxon did not occur when these carrier erythrocytes contained no phosphotriesterase. These in vitro studies suggest that carrier erythrocytes may be developed as an approach for the prophylactic and therapeutic antagonism of organophosphorus intoxication.

Enzymes as pretreatment drugs for organophosphate toxicity.

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.