Efficacy of HD201 vs Referent Trastuzumab in Patients With ERBB2-Positive Breast Cancer Treated in the Neoadjuvant Setting: A Multicenter Phase 3 Randomized Clinical Trial.

Importance: The drug HD201 is a biosimilar candidate for breast cancer treatment as the reference trastuzumab. Objective: To compare the efficacy of HD201 with referent trastuzumab. Design, Setting, and Participants: This randomized clinical trial (TROIKA) included 502 women with ERBB2-positive early breast cancer treated with either HD201 or referent trastuzumab. It was conducted across 70 centers in 12 countries, including Western and Eastern Europe and Asian countries. Randomization was stratified by tumor hormone receptor status, clinical stage, and geographic region of recruitment. This analysis was conducted on February 12, 2021, after the completion of the adjuvant phase at a median of 31 months (IQR, 28-33 months) of follow-up. Interventions: Patients with ERBB2-positive early breast cancer were randomly assigned to receive HD201 or referent trastuzumab in the neoadjuvant setting for 8 cycles, concurrently with 4 cycles of docetaxel, which was followed by 4 cycles of epirubicin and cyclophosphamide. Patients then underwent surgery, which was followed by treatment with 10 cycles of adjuvant HD201 or referent trastuzumab. Main Outcome and Measures: The primary end point was the total pathological complete response (tpCR) assessed after neoadjuvant treatment. Equivalence was concluded if the 95% CI of the absolute difference in tpCR between arms in the per-protocol set was within the margin of more or less than 15%. Other objectives included the breast pathological complete response, overall response, event-free and overall survival, safety, pharmacokinetics, and immunogenicity. Results: A total of 502 female patients (mean [range] age, 53 [26-82] years) were randomized to receive either HD201 or referent trastuzumab, and 474 (94.2%) were eligible for inclusion in the per-protocol set. The baseline characteristics were well balanced between the 2 arms; 195 tumors (38.8%) were hormone receptor-negative , and 213 patients (42.4%) had clinical stage III disease. The tpCR rates were 45% and 48.7% for HD201 and referent trastuzumab, respectively. The difference between the 2 groups was not significant at -3.8% (95% CI, -12.8% to 5.4%) and fell within the predefined equivalence margins. The ratio of the tpCR rates between the 2 arms was 0.92 (95% CI, 0.76 to 1.12). A total of 433 patients (86.1%) presented with 2232 treatment-emergent adverse events of special interest for trastuzumab during the entire treatment period, with 220 (88.0%) and 213 (84.5%) patients in the HD201 and referent trastuzumab groups, respectively. Conclusions and Relevance: The results of this randomized clinical trial found that HD201 demonstrated equivalence to referent trastuzumab in terms of efficacy for the end point of tpCR, with a similar safety profile. Trial Registration: ClinicalTrials.gov Identifier: NCT03013504.

Bioengineering potato plants to produce benzylglucosinolate for improved broad-spectrum pest and disease resistance.

In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development.

NITD-688, a pan-serotype inhibitor of the dengue virus NS4B protein, shows favorable pharmacokinetics and efficacy in preclinical animal models.

Dengue virus (DENV) is a mosquito-borne flavivirus that poses a threat to public health, yet no antiviral drug is available. We performed a high-throughput phenotypic screen using the Novartis compound library and identified candidate chemical inhibitors of DENV. This chemical series was optimized to improve properties such as anti-DENV potency and solubility. The lead compound, NITD-688, showed strong potency against all four serotypes of DENV and demonstrated excellent oral efficacy in infected AG129 mice. There was a 1.44-log reduction in viremia when mice were treated orally at 30 milligrams per kilogram twice daily for 3 days starting at the time of infection. NITD-688 treatment also resulted in a 1.16-log reduction in viremia when mice were treated 48 hours after infection. Selection of resistance mutations and binding studies with recombinant proteins indicated that the nonstructural protein 4B is the target of NITD-688. Pharmacokinetic studies in rats and dogs showed a long elimination half-life and good oral bioavailability. Extensive in vitro safety profiling along with exploratory rat and dog toxicology studies showed that NITD-688 was well tolerated after 7-day repeat dosing, demonstrating that NITD-688 may be a promising preclinical candidate for the treatment of dengue.

Botanical from the medicinal spice, Piper capense is safe as demonstrated by oral acute and subchronic toxicity investigations.

Piper capense Linn is a plant used in Cameroon to treat cancer and several other diseases such as urinary tract disorder, fever, stomach-ache and to improve appetite. The methanol extract of Piper capense has been reported for its antiproliferative activity towards several human cancer cell lines. The aim of this work was to evaluate the acute and subchronic oral toxicities of a methanol extract from P. capense fruits on rats. The acute oral toxicity assay was carried out by administration of a single dose of 5000 mg/kg body weight of methanol extract of the Piper capense to five female rats, after which the behavior of the animals and the number of deaths were noted after 48 h. The animals were then kept for observation for 14 days. On the 15th day, the rats were sacrificed and macroscopic observation of the organs was made. Concerning the subchronic toxicity study, the rats composed of males and females received three doses (250, 500 and 1000 mg/kg body weight/day) for a period of 28 days by oral gavage. General animal behavior, food intake, weight gain, organ weights, haematological parameters, serum, and urinary biochemical parameters, and histological sections of liver and kidneys, were evaluated. Methanol extract from the Piper capense fruits did not cause any death in rats that were administered a single dose of 5000 mg/kg body weight of extract and therefore, the letal dose 50 (LD50) of the extract is greater than 5000 mg/kg body weight. Subchronic administration of the methanol extract of Piper capense fruits showed significant variations (P > 0.05) after analysis of certain biochemical parameters: serum urea, urinary urea, alanine aminotransferase (ALAT), aspartate aminotransferase, (ASAT), serum protein; in both male and female rats that received the dose of 1000 mg/kg body weight/day. No major signs of toxicity were observed in the liver and kidneys of animals after analysis of the histological sections performed. Beside, some signs of toxicity were observed, including cell lysis and inflammation on the liver and kidney organs at a dose of 1000 mg/kg body weight/day. Finally, the methanol extract of Piper capense fruits is safe at lower doses, but could cause some damages at doses as high as 1000 mg/kg body weight/day. Consequently, it should be taken with caution when used in therapy.

In Situ Impedance Analysis of Oxygen Exchange on Growing La0.6Sr0.4CoO3-δ Thin Films.

The further development of solid oxide fuel and electrolysis cells (SOFC/SOEC) strongly relies on research activities dealing with electrode materials. Recent studies showed that under operating conditions many perovskite-type oxide electrodes are prone to changes of their surface composition, leading to severe changes of their electrochemical performance. This results in a large scatter of data in literature and complicates comparison of materials. Moreover, little information is available on the potentially excellent properties of surfaces immediately after preparation, that is, before any degradation by exposure to other gas compositions or temperature changes. Here, we introduce in situ impedance spectroscopy during pulsed laser deposition (IPLD) as a new method for electrochemical analysis of mixed ionic and electronic conducting (MIEC) thin films during growth. First, this approach can truly reveal the properties of as-prepared MIEC electrode materials, since it avoids any alterations of their surface between preparation and investigation. Second, the measurements during growth give information on the thickness dependence of film properties. This technique is applied to La0.6Sr0.4CoO3-δ (LSC), one of the most promising SOFC/SOEC oxygen electrode material. From the earliest stages of LSC film deposition on yttria-stabilized zirconia (YSZ) to a fully grown thin film of 100 nm thickness, data are gained on the oxygen exchange kinetics and the defect chemistry of LSC. A remarkable reproducibility is found in repeated film growth experiments, not only for the bulk related chemical capacitance but also for the surface related polarization resistance (±10%). Polarization resistances of as-prepared LSC films are extraordinarily low (2.0 Ω cm2 in 40 µbar O2 at 600 °C). LSC films on YSZ and on La0.95Sr0.05Ga0.95Mg0.05O3-δ (LSGM) single crystals exhibit significantly different electrochemical properties, possibly associated with the tensile strain of LSC on LSGM.

How To Get Mechanistic Information from Partial Pressure-Dependent Current-Voltage Measurements of Oxygen Exchange on Mixed Conducting Electrodes.

The oxygen incorporation and evolution reaction on mixed conducting electrodes of solid oxide fuel or electrolysis cells involves gas molecules as well as ionic and electronic point defects in the electrode. The defect concentrations depend on the gas phase and can be modified by the overpotential. These interrelationships make a mechanistic analysis of partial pressure-dependent current-voltage experiments challenging. In this contribution it is described how to exploit this complex situation to unravel the kinetic roles of surface adsorbates and electrode point defects. Essential is a counterbalancing of oxygen partial pressure and dc electrode polarization such that the point defect concentrations in the electrode remain constant despite varying the oxygen partial pressure. It is exemplarily shown for La0.6Sr0.4FeO3-δ (LSF) thin film electrodes on yttria-stabilized zirconia how mechanistically relevant reaction orders can be obtained from current-voltage curves, measured in a three-electrode setup. This analysis strongly suggests electron holes as the limiting defect species for the oxygen evolution on LSF and reveals the dependence of the oxygen incorporation rate on the oxygen vacancy concentration. A virtual independence of the reaction rate from the oxygen partial pressure was empirically found for moderate oxygen pressures. This effect, however, arises from a counterbalancing of defect and adsorbate concentration changes.

Voltage and partial pressure dependent defect chemistry in (La,Sr)FeO3-δ thin films investigated by chemical capacitance measurements.

La0.6Sr0.4FeO3-δ (LSF) thin films of different thickness were prepared by pulsed laser deposition on yttria stabilized zirconia (YSZ) and characterized by using three electrode impedance spectroscopy. Electrochemical film capacitance was analyzed in relation to oxygen partial pressure (0.25 mbar to 1 bar), DC polarization (0 m to -600 m) and temperature (500 to 650 °C). For most measurement parameters, the chemical bulk capacitance dominates the overall capacitive properties and the corresponding defect chemical state depends solely on the oxygen chemical potential inside the film, independent of atmospheric oxygen pressure and DC polarization. Thus, defect chemical properties (defect concentrations and defect formation enthalpies) could be deduced from such measurements. Comparison with LSF defect chemical bulk data from the literature showed good agreement for vacancy formation energies but suggested larger electronic defect concentrations in the films. From thickness-dependent measurements at lower oxygen chemical potentials, an additional capacitive contribution could be identified and attributed to the LSF|YSZ interface. Deviations from simple chemical capacitance models at high pressures are most probably due to defect interactions.

Imidazolopiperazines Kill both Rings and Dormant Rings in Wild-Type and K13 Artemisinin-Resistant Plasmodium falciparum In Vitro.

Artemisinin (ART) resistance has spread through Southeast Asia, posing a serious threat to the control and elimination of malaria. ART resistance has been associated with mutations in the Plasmodium falciparum kelch-13 (Pfk13) propeller domain. Phenotypically, ART resistance is defined as delayed parasite clearance in patients due to the reduced susceptibility of early ring-stage parasites to the active metabolite of ART dihydroartemisinin (DHA). Early rings can enter a state of quiescence upon DHA exposure and resume growth in its absence. These quiescent rings are referred to as dormant rings or DHA-pretreated rings (here called dormant rings). The imidazolopiperazines (IPZ) are a novel class of antimalarial drugs that have demonstrated efficacy in early clinical trials. Here, we characterized the stage of action of the IPZ GNF179 and evaluated its activity against rings and dormant rings in wild-type and ART-resistant parasites. Unlike DHA, GNF179 does not induce dormancy. We show that GNF179 is more rapidly cidal against schizonts than against ring and trophozoite stages. However, with 12 h of exposure, the compound effectively kills rings and dormant rings of both susceptible and ART-resistant parasites within 72 h. We further demonstrate that in combination with ART, GNF179 effectively prevents recrudescence of dormant rings, including those bearing pfk13 propeller mutations.

Development of a Cytopathic Effect-Based Phenotypic Screening Assay against Cryptosporidium.

Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum ( Cp) and Cryptosporidium hominis ( Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs and confirming many previously known anti- Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, and homogeneous gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials.

The Chemical Evolution of the La0.6Sr0.4CoO3-δ Surface Under SOFC Operating Conditions and Its Implications for Electrochemical Oxygen Exchange Activity.

Owing to its extraordinary high activity for catalysing the oxygen exchange reaction, strontium doped LaCoO3 (LSC) is one of the most promising materials for solid oxide fuel cell (SOFC) cathodes. However, under SOFC operating conditions this material suffers from performance degradation. This loss of electrochemical activity has been extensively studied in the past and an accumulation of strontium at the LSC surface has been shown to be responsible for most of the degradation effects. The present study sheds further light onto LSC surface changes also occurring under SOFC operating conditions. In-situ near ambient pressure X-ray photoelectron spectroscopy measurements were conducted at temperatures between 400 and 790 °C. Simultaneously, electrochemical impedance measurements were performed to characterise the catalytic activity of the LSC electrode surface for O2 reduction. This combination allowed a correlation of the loss in electro-catalytic activity with the appearance of an additional La-containing Sr-oxide species at the LSC surface. This additional Sr-oxide species preferentially covers electrochemically active Co sites at the surface, and thus very effectively decreases the oxygen exchange performance of LSC. Formation of precipitates, in contrast, was found to play a less important role for the electrochemical degradation of LSC.

A comparative transcriptomic analysis of replicating and dormant liver stages of the relapsing malaria parasite Plasmodium cynomolgi.

Plasmodium liver hypnozoites, which cause disease relapse, are widely considered to be the last barrier towards malaria eradication. The biology of this quiescent form of the parasite is poorly understood which hinders drug discovery. We report a comparative transcriptomic dataset of replicating liver schizonts and dormant hypnozoites of the relapsing parasite Plasmodium cynomolgi. Hypnozoites express only 34% of Plasmodium physiological pathways, while 91% are expressed in replicating schizonts. Few known malaria drug targets are expressed in quiescent parasites, but pathways involved in microbial dormancy, maintenance of genome integrity and ATP homeostasis were robustly expressed. Several transcripts encoding heavy metal transporters were expressed in hypnozoites and the copper chelator neocuproine was cidal to all liver stage parasites. This transcriptomic dataset is a valuable resource for the discovery of vaccines and effective treatments to combat vivax malaria.

Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen.

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

The Natural Product Cavinafungin Selectively Interferes with Zika and Dengue Virus Replication by Inhibition of the Host Signal Peptidase.

Flavivirus infections by Zika and dengue virus impose a significant global healthcare threat with no US Food and Drug Administration (FDA)-approved vaccination or specific antiviral treatment available. Here, we present the discovery of an anti-flaviviral natural product named cavinafungin. Cavinafungin is a potent and selectively active compound against Zika and all four dengue virus serotypes. Unbiased, genome-wide genomic profiling in human cells using a novel CRISPR/Cas9 protocol identified the endoplasmic-reticulum-localized signal peptidase as the efficacy target of cavinafungin. Orthogonal profiling in S. cerevisiae followed by the selection of resistant mutants pinpointed the catalytic subunit of the signal peptidase SEC11 as the evolutionary conserved target. Biochemical analysis confirmed a rapid block of signal sequence cleavage of both host and viral proteins by cavinafungin. This study provides an effective compound against the eukaryotic signal peptidase and independent confirmation of the recently identified critical role of the signal peptidase in the replicative cycle of flaviviruses.

Real-time impedance monitoring of oxygen reduction during surface modification of thin film cathodes.

Improvement of solid oxide fuel cells strongly relies on the development of cathode materials with high catalytic activity for the oxygen reduction reaction. Excellent activity was found for perovskite-type oxides such as La1-xSrxCoO3-δ (LSC), but performance degradation, probably caused by surface composition changes, hinders exploitation of the full potential of LSC. This study reveals that the potentially very high activity of the LSC surface can be traced back to few very active sites. Already tiny amounts of SrO, for example, 4% of a monolayer, deposited on an LSC surface, lead to severe deactivation. Co, on the other hand, causes (re-)activation, suggesting that active sites are strongly related to Co being present at the surface. These insights could be gained by a novel method to measure changes of the electrochemical performance of thin film electrodes in situ, while modifying their surface: impedance spectroscopy measurements during deposition of well-defined fractions of monolayers of Sr-, Co- and La-oxides by single laser pulses in a pulsed laser deposition chamber.

Discovery of 2-oxopiperazine dengue inhibitors by scaffold morphing of a phenotypic high-throughput screening hit.

A series of 2-oxopiperazine derivatives were designed from the pyrrolopiperazinone cell-based screening hit 4 as a dengue virus inhibitor. Systematic investigation of the structure-activity relationship (SAR) around the piperazinone ring led to the identification of compound (S)-29, which exhibited potent anti-dengue activity in the cell-based assay across all four dengue serotypes with EC50<0.1µM. Cross-resistant analysis confirmed that the virus NS4B protein remained the target of the new oxopiperazine analogs obtained via scaffold morphing from the HTS hit 4.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes.

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

The Chemical Capacitance as a Fingerprint of Defect Chemistry in Mixed Conducting Oxides.

The oxygen stoichiometry of mixed conducting oxides depends on the oxygen chemical potential and thus on the oxygen partial pressure in the gas phase. Also voltages may change the local oxygen stoichiometry and the amount to which such changes take place is quantified by the chemical capacitance of the sample. Impedance spectroscopy can be used to probe this chemical capacitance. Impedance measurements on different oxides ((La,Sr)FeO3-δ = LSF, Sr(Ti,Fe)O3-δ = STF, and Pb(Zr,Ti)O3 = PZT) are presented, and demonstrate how the chemical capacitance may affect impedance spectra in different types of electrochemical cells. A quantitative analysis of the spectra is based on generalized equivalent circuits developed for mixed conducting oxides by J. Jamnik and J. Maier. It is discussed how defect chemical information can be deduced from the chemical capacitance.