Preclinical efficacy of hK2 targeted [177Lu]hu11B6 for prostate cancer theranostics

Androgen ablating drugs increase life expectancy in men with metastatic prostate cancer, but resistance inevitably develops. In a majority of these recurrent tumors, the androgen axis is reactivated in the form of increased androgen receptor (AR) expression. Targeting proteins that are expressed as a down-stream effect of AR activity is a promising rationale for management of this disease. The humanized IgG1 antibody hu11B6 internalizes into prostate and prostate cancer (PCa) cells by binding to the catalytic cleft of human kallikrein 2 (hK2), a prostate specific enzyme governed by the AR-pathway. In a previous study, hu11B6 conjugated with Actinium-225 (225Ac), a high linear energy transfer (LET) radionuclide, was shown to generate an AR-upregulation driven feed-forward mechanism that is believed to enhance therapeutic efficacy. We assessed the efficacy of hu11B6 labeled with a low LET beta-emitter, Lutetium-177 (177Lu) and investigated whether similar tumor killing and AR-enhancement is produced. Moreover, single-photon emission computed tomography (SPECT) imaging of 177Lu is quantitatively accurate and can be used to perform treatment planning. [177Lu]hu11B6 therefore has significant potential as a theranostic agent. Materials and Methods: Subcutaneous PCa xenografts (LNCaP s.c.) were grown in male mice. Biokinetics at 4-336 h post injection and uptake as a function of the amount of hu11B6 injected at 72 h were studied. Over a 30 to 120-day treatment period the therapeutic efficacy of different activities of [177Lu]hu11B6 were assessed by volumetric tumor measurements, blood cell counts, molecular analysis of the tumor as well as SPECT/CT imaging. Organ specific mean absorbed doses were calculated, using a MIRD-scheme, based on biokinetic data and rodent specific S-factors from a modified MOBY phantom. Tumor tissues of treated xenografts were immunohistochemically (IHC) stained for Ki-67 (proliferation) and AR, SA-β-gal activity (senescence) and analyzed by digital autoradiography (DAR). Results: Organ-to-blood and tumor-to-blood ratios were independent of hu11B6 specific activity except for the highest amount of antibody (150 µg). Tumor accumulation of [177Lu]hu11B6 peaked at 168 h with a specific uptake of 29 ± 9.1 percent injected activity per gram (%IA/g) and low accumulation in normal organs except in the submandibular gland (15 ± 4.5 %IA/g), attributed to a cross-reaction with mice kallikreins in this organ, was seen. However, SPECT imaging with therapeutic amounts of [177Lu]hu11B6 revealed no peak in tumor accumulation at 7 d, probably due to cellular retention of 177Lu and decreasing tumor volumes. For [177Lu]hu11B6 treated mice, tumor decrements of up to 4/5 of the initial tumor volume and reversible myelotoxicity with a nadir at 12 d were observed after a single injection. Tumor volume reduction correlated with injected activity and the absorbed dose. IHC revealed retained expression of AR throughout treatment and that Ki-67 staining reached a nadir at 9-14 d which coincided with high SA- β-gal activity (14 d). Quantification of nuclei staining showed that Ki-67 expression correlated negatively with activity uptake. AR expression levels in cells surviving therapy compared to previous timepoints and to controls at 30 d were significantly increased (p = 0.017). Conclusions: This study shows that hu11B6 labeled with the low LET beta-emitting radionuclide 177Lu can deliver therapeutic absorbed doses to prostate cancer xenografts with transient hematological side-effects. The tumor response correlated with the absorbed dose both on a macro and a small scale dosimetric level. Analysis of AR staining showed that AR protein levels increased late in the study suggesting a therapeutic mechanism, a feed forward mechanism coupled to AR driven response to DNA damage or clonal lineage selection, similar to that reported in high LET alpha-particle therapy using 225Ac labeled hu11B6, however emerging at a later timepoint

Preparation of catalytic substrates with ordered size of iron nanoparticles for production carbon nanotubes

Canberra, ACT, Australi

Reproducible model development in the Cardiac Electrophysiology Web Lab

The modelling of the electrophysiology of cardiac cells is one of the most mature areas of systems biology. This extended concentration of research effort brings with it new challenges, foremost among which is that of choosing which of these models is most suitable for addressing a particular scientific question. In a previous paper, we presented our initial work in developing an online resource for the characterisation and comparison of electrophysiological cell models in a wide range of experimental scenarios. In that work, we described how we had developed a novel protocol language that allowed us to separate the details of the mathematical model (the majority of cardiac cell models take the form of ordinary differential equations) from the experimental protocol being simulated. We developed a fully-open online repository (which we termed the Cardiac Electrophysiology Web Lab) which allows users to store and compare the results of applying the same experimental protocol to competing models. In the current paper we describe the most recent and planned extensions of this work, focused on supporting the process of model building from experimental data. We outline the necessary work to develop a machine-readable language to describe the process of inferring parameters from wet lab datasets, and illustrate our approach through a detailed example of fitting a model of the hERG channel using experimental data. We conclude by discussing the future challenges in making further progress in this domain towards our goal of facilitating a fully reproducible approach to the development of cardiac cell models

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Dehydropolymerization of H3B·NMeH2 Using a [Rh(DPEphos)]+ Catalyst : The Promoting Effect of NMeH2

[Rh(κ2-PP-DPEphos){η2η2-H2B(NMe3)(CH2)2tBu}][BArF4] acts as an effective precatalyst for the dehydropolymerization of H3B·NMeH2 to form N-methylpolyaminoborane (H2BNMeH)n. Control of polymer molecular weight is achieved by variation of precatalyst loading (0.1-1 mol %, an inverse relationship) and use of the chain-modifying agent H2: with Mn ranging between 5 500 and 34 900 g/mol and between 1.5 and 1.8. H2 evolution studies (1,2-F2C6H4 solvent) reveal an induction period that gets longer with higher precatalyst loading and complex kinetics with a noninteger order in [Rh]TOTAL. Speciation studies at 10 mol % indicate the initial formation of the amino-borane bridged dimer, [Rh2(κ2-PP-DPEphos)2(μ-H)(μ-H2BN=HMe)][BArF4], followed by the crystallographically characterized amidodiboryl complex [Rh2(cis-κ2-PP-DPEphos)2(σ,μ-(H2B)2NHMe)][BArF4]. Adding ∼2 equiv of NMeH2 in tetrahydrofuran (THF) solution to the precatalyst removes this induction period, pseudo-first-order kinetics are observed, a half-order relationship to [Rh]TOTAL is revealed with regard to dehydrogenation, and polymer molecular weights are increased (e.g., Mn = 40 000 g/mol). Speciation studies suggest that NMeH2 acts to form the precatalysts [Rh(κ2-DPEphos)(NMeH2)2][BArF4] and [Rh(κ2-DPEphos)(H)2(NMeH2)2][BArF4], which were independently synthesized and shown to follow very similar dehydrogenation kinetics, and produce polymers of molecular weight comparable with [Rh(κ2-PP-DPEphos){ η2-H2B(NMe3)(CH2)2tBu}][BArF4], which has been doped with amine. This promoting effect of added amine in situ is shown to be general in other cationic Rh-based systems, and possible mechanistic scenarios are discussed

Molecular differentiation of the Murraya paniculata Complex (Rutaceae: Aurantioideae: Aurantieae)

Background: Orange jasmine has a complex nomenclatural history and is now known as Murraya paniculata (L.) Jack. Our interest in this common ornamental stemmed from the need to resolve its identity and the identities of closely related taxa as hosts of the pathogen 'Candidatus Liberibacter asiaticus' and its vector Diaphorina citri. Understanding these microbe-vector-plant relationships has been hampered by taxonomic confusion surrounding Murraya at both the generic and specific levels. Results: To resolve the taxonomic uncertainty, six regions of the maternally-inherited chloroplastal genome and part of the nuclear-encoded ITS region were amplified from 85 accessions of Murraya and Merrillia using the polymerase chain reaction (PCR). Clustering used maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI). Chronograms were produced for molecular dating, and to test the monophyly of Murraya rigorously, using selected accessions of Murraya and 26 accessions of the Rutaceae and Simarubaceae. Sequence data from the ITS and chloroplastal regions suggest that Murraya paniculata (sensu (Swingle WT and Reece CR, The Citrus Industry, p. 190-430, 1967)) can be separated into four distinct but morphologically somewhat cryptic taxa: Murraya paniculata (sensu (Mabberley DJ, Taxon 65:366-371, 2016)), M. elongata, M. sumatrana and M. lucida. In addition, Murraya omphalocarpa was identified as a putative hybrid of M. paniculata and M. lucida with two geographically isolated nothovarieties representing reciprocal crosses. Murraya is monophyletic, and molecular dating suggests that it diverged from Merrillia during the Miocene (23-5 Ma) with this Murraya group speciating and dispersing during the Middle Miocene onwards. Conclusions: The accessions from Asia and Australasia used in this study grouped into biogeographical regions that match herbarium specimen records for the taxa that suggest natural allopatric distributions with limited overlap and hybridity. Murraya paniculata has been distributed around the world as an ornamental plant. The division of the Murraya paniculata complex into four species with a rare hybrid also confirms morphological studies

Rapid Characterization of hERG Channel Kinetics II: Temperature Dependence

© 2019 Biophysical Society Ion channel behavior can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing, inactivating, and recovery) are commonly represented with Q10 coefficients or an Eyring relationship. In this article, we assess the validity of these representations by characterizing channel kinetics at multiple temperatures. We focus on the human Ether-à-go-go-Related Gene (hERG) channel, which is important in drug safety assessment and commonly screened at room temperature so that results require extrapolation to physiological temperature. In Part I of this study, we established a reliable method for high-throughput characterization of hERG1a (Kv11.1) kinetics, using a 15-second information-rich optimized protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using Chinese hamster ovary cells overexpressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, with temperature control. We characterize the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37°C and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients; it broadly follows a generalized, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol dependent. Our results show that a direct fit using our 15-s protocol best represents hERG kinetics at any given temperature and suggests that using the Generalized Eyring theory is preferable if no experimental data are available to derive model parameters at a given temperature

‘Scots and Scabs from North-by-Tweed’:Undesirable Scottish Migrants in Seventeenth- and Early Eighteenth-Century England

While very prominent in the contemporary world, anxiety about the potentially negative impact that immigrants might have on their host communities has deep historical roots. In a British context, such fears were particularly heightened following the regal union of 1603 when large numbers of Scots began settling in England. This article offers a fresh perspective on these issues by exploring the experiences and reception of poor, deviant or otherwise ‘undesirable’ Scottish migrants to England during the seventeenth and eighteenth centuries. Focusing in particular on chapmen, vagrants and criminals, it suggests that, while in general Scots were able to integrate relatively easily into English society, there existed an unwelcome subset surviving by dubious means. Though not usually attracting unduly severe treatment on account of their nationality, these unwelcome migrants had a disproportionate effect on English perceptions of and attitudes towards the broader cohort of Scottish migrants in their midst