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A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation
No full textPlasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches
Structural Analysis Reveals that the Cytokine IL-17F Forms a Homodimeric Complex with Receptor IL-17RC to Drive IL-17RA-Independent Signaling
No full textIL-17, a crucial cytokine for chronic inflammatory diseases, forms a heteromeric complex with IL-17RA and IL-17RC receptors for signaling. Goepfert et al. determine the structure of human IL-17F bound to IL-17RC and reveal a homodimeric assembly that contrasts with the prevailing signaling paradigm and suggests IL-17RA-independent roles for IL-17RC.
Interleukin-17A (IL-17A), IL-17F, and IL-17A/F heterodimers are key cytokines of the innate and adaptive immune response. Dysregulation of the IL-17 pathway contributes to immune pathology, and it is therefore important to elucidate the molecular mechanisms that govern IL-17 recognition and signaling. The receptor IL-17RC is thought to act in concert with IL-17RA to transduce IL-17A-, IL-17F-, and IL-17A/F-mediated signals. We report the crystal structure of the extracellular domain of human IL-17RC in complex with IL-17F. In contrast to the expected model, we found that IL-17RC formed a symmetrical 2:1 complex with IL-17F, thus competing with IL-17RA for cytokine binding. Using biophysical techniques, we showed that IL-17A and IL-17A/F also form 2:1 complexes with IL-17RC, suggesting the possibility of IL-17RA-independent IL-17 signaling pathways. The crystal structure of the IL-17RC:IL-17F complex provides a structural basis for IL-17F signaling through IL-17RC, with potential therapeutic applications for respiratory allergy and inflammatory bowel diseases
A murine model of meningeal inflammation and subpial demyelination identifies an anti-MOG independent mechanism of cortical injury that is inhibited by Siponimod therapy
No full textSubpial demyelination is a specific hallmark of multiple sclerosis (MS) and a correlate of
disease progression and cognitive decline. Although the mechanism(s) that mediate
pathogenesis in this compartment remain unclear, it has been speculated that inflammation in
the overlying meninges may be associated with sub-pial injury. We have previously shown that
proteolipid-primed Th17 cells adoptively transferred into SJL/J recipient mice induce
Experimental Autoimmune Encephalomyelitis (A/T SJL/J EAE) accompanied by extensive
stromal cell remodelling and accumulation of lymphocytes in the brain meninges. Here we
show that meningeal inflammation in this model overlays areas of subpial cortical
demyelination associated with microglial/macrophage activation, disruption of the glial
limitans and evidence of an oxidative stress response. These pathological features were
observed even in the absence of measurable anti-MOG IgM or IgG antibodies. We took
advantage of this model to test potential mechanisms of action of BAF312 (siponimod), a
selective sphingosine 1-phosphate (S1P) receptor1,5 modulator. BAF312 treatment
significantly ameliorated clinical manifestations of EAE concomitant with diminished
meningeal inflammation and subpial pathology and as well as a selective reduction in Th17
cell accumulation in the central nervous system (CNS). We conclude that Th17 cells rather
than anti-MOG antibodies are critically required for meningeal inflammation and cortical
pathology in A/T SJL/J EAE. Moreover BAF312, a promising MS therapeutic, ameliorates the
clinical and pathological features of this model perhaps through its affect on CNS-resident
Th17 cells
Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis
No full textThe lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery
Tg.rasH2 mouse model for Assessing Carcinogenic Potential of Pharmaceuticals: Industry Survey and Recommendations
No full textThe Tg.rasH2 mouse was developed as a model for the alternative carcinogenicity testing. As an alternative to traditional 2-year mouse bioassay, this model has found extensive use in support of biopharmaceutical drug development over the last few decades. It has the potential of improving quality, timeliness, reduction of animal numbers, and in some instances allow expedient decision making regarding the potential for human carcinogenicity. Despite the increased use of the Tg.rasH2 model, there has been no systematic survey of current practices in the design, interpretation of results from the bioassay, and global health authority perspectives. Therefore, the aim of this work was to poll the pharmaceutical industry on study design practices used in the dose range finding and definitive 6-month Tg.rasH2 study, and on results relative to the ongoing revisions to ICH S1. Participants in the survey are member companies of IQ DruSafe. The survey results provide very useful insights in study design and interpretation. Importantly, the results identified several key opportunities for reducing animal use and increasing the value of testing for potential human carcinogenicity using this model
Fluorine NMR functional screening: from purified enzymes to human intact living cells
No full textThe substrate- or cofactor-based fluorine NMR screening, also known as n-FABS (n fluorine atoms for biochemical screening), represents a powerful method for performing a direct functional assay in the search of inhibitors or enhancers of an enzymatic reaction. Although it suffers from the intrinsic low sensitivity compared to other biophysical techniques usually applied in functional assays, it has some distinctive features that makes it appealing for tackling complex chemical and biological systems. Its strengths are represented by the easy set-up, robustness, flexibility, lack of signal interference and rich information content resulting in the identification of bona fide inhibitors and reliable determination of their inhibitory strength. The versatility of the n-FABS allows its application to either purified enzymes, cell lysates or intact living cells. The principles, along with theoretical, technical and practical aspects, of the methodology are discussed. Furthermore, several applications of the technique to pharmaceutical projects are presented
Assessment of the anti-CD40 antibody iscalimab in patients with primary Sjögren’s syndrome: a multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study
No full textBackground Primary Sjögren’s syndrome is an autoimmune disease that presents as dryness of the mouth and eyes due to impairment of the exocrine glands. To our knowledge, no systemic therapies for primary Sjögren’s syndrome have shown efficacy. CD40–CD154-mediated T cell–B cell interactions in primary Sjögren’s syndrome contribute to aberrant lymphocyte activation in inflamed tissue, leading to sialadenitis and other tissue injury. Therefore, we investigated the safety and preliminary efficacy of iscalimab (CFZ533), a novel anti-CD40 monoclonal antibody, in patients with primary Sjögren’s syndrome.
Methods This multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study took place at ten investigational sites across Europe (UK, n=4; Germany, Switzerland, and Hungary, n=1 each) and the USA (n=3). Eligible patients were aged 18–75 years and fulfilled the 2002 American European consensus group diagnostic classification criteria for primary Sjögren’s syndrome. In the double-blind phase of the trial, patients were randomly assigned (2:1) via computer-generated unique randomisation numbers to receive subcutaneous iscalimab (3 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 1) or intravenous iscalimab (10 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 2). Randomisation was stratified according to baseline intake of oral corticosteroids. At week 12, patients in both cohorts received open-label iscalimab (same dose and route) for 12 weeks. The primary objectives of the study were to assess the safety, tolerability, and efficacy of multiple doses of iscalimab in the two sequential dose cohorts. Safety and tolerability were assessed by adverse events and efficacy of iscalimab versus placebo was assessed by clinical disease activity, as measured by the change in European League Against Rheumatism Sjögren’s syndrome disease activity index (ESSDAI) score after 12 weeks of treatment. Analyses were done on a per-protocol basis. The trial was registered with ClinicalTrials.gov, NCT02291029.
Findings Between Oct 22, 2014, and June 28, 2016, we assessed 82 patients for eligibility (25 for cohort 1 and 57 for cohort 2). 38 patients were excluded because of ineligibility. In cohort 1, 12 patients were randomly assigned to receive either 3 mg/kg doses of iscalimab (n=8) or placebo (n=4), and in cohort 2, 32 patients were randomly assigned to receive either intravenous 10 mg/kg doses of iscalimab (n=21) or placebo (n=11). Adverse events were similar between iscalimab treatment groups and placebo groups, with adverse events occurring in all patients in cohort 1, and in 52% and 64% of the iscalimab and placebo groups, respectively, in cohort 2. Two serious adverse events were reported (one case of bacterial conjunctivitis in cohort 1 and one case of atrial fibrillation in cohort 2), which were unrelated to treatment with iscalimab. Intravenous treatment with iscalimab resulted in a mean reduction of 5·21 points (95% CI 0·96–9·46; one-sided p=0·0090) in ESSDAI score compared with placebo. There was no signficiant difference in ESSDAI score between subcutaneous iscalimab and placebo.
Interpretation To our knowledge, this is the first randomised, placebo-controlled proof-of-concept study of a new investigational drug for primary Sjögren’s syndrome that indicates preliminary efficacy. Our data suggest a role of CD40–CD154 interactions in primary Sjögren’s syndrome pathology and the therapeutic potential for CD40 blockade in this disease should be investigated further
Deep learning for continuous manufacturing of pharmaceutical solid dosage form
No full textContinuous Manufacturing (CM) of pharmaceutical drug products is a new approach within the pharmaceutical industry. In the presented paper, a GMP continuous wet granulation line for production of solid dosage forms was investigated. The line was composed of the subsequent continuous unit: operations feeding – twin-screw wet-granulation – fluid-bed drying – sieving and tableting. The formulation of a commercial entity was selected for this study. Several critical process parameters were evaluated in order to probe the process and to characterize the impact on quality attributes. Seven critical process parameters have been selected after a risk analysis: API and excipient mass flows of the two feeders, liquid feed rate and rotation speed of the extruder and rotation speed, temperature and airflow of the dryer. Eight quality attributes were controlled in real time by Process Analytical Technologies (PAT): API content after blender, after dryer, in tablet press feed frame and of tablet, LOD after dryer and PSD after dryer (three PSD parameters: x10 x50 x90). The process parameter values were changed during production in order to detect the impact on the quality of the final product. The deep learning techniques have been used in order to predict the quality attribute (output) with the process parameters (input). The use of deep learning reduces the noise and simplify the data interpretation for a better process understanding. After optimization, three hidden layers neural network were selected with 6 hidden neurons. The activation function ReLU (Rectified Linear Unit) and the ADAM optimizer were used with 2500 epochs (number of learning cycle). API contents, PSD values and LOD values were estimated with an error of calibration lower than 10%. The level of error allow an adequate process monitoring by DNN and we have proven that the main critical process parameters can be identified at a higher levelof process understanding. The synergy between PAT and process data science creates a superior monitoring framework of the continuous manufacturing line and increase the knowledge of this innovative production line and the products that it make
PhD Thesis: Technologies for Vascularized Skin Equivalents to Study Cutaneous Wound Healing in Vitro
No full textPhD Thesis, Lisa Kiesewetter, "Technische Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades Dr.-Ing." University of Erlangen-Nürnberg, Germany.
Some of the work mentioned in the thesis are based on a Master Service Agreement of NIBR with the Fraunhofer Institute, dated August 14, 2015, aimed at a publication for the establishment of an in vitro skin model (exchange of protocols, reagents, positive control recombinant growth factor IGF-1). Due to lack of efficacy of the tested agents in the models, the data have not been submitted for publication. The NIBR contributions are mentioned on the following sections of the thesis:
Methods: pp. 66-67
Results: pp. 88-94
Discussion: pp. 151-152
Appendix: p. 203
Acknowledgements: p. 211
The two NVS employees mentioned below are acknowledged in the thesis, but they are not authors.
The models presented in this thesis should provide new, sophisticated alternatives for animal experiments in the field of research on cutaneous wound healing. Novel active pharmaceutical ingredients can be studied employing wound models meeting the specific experimental needs and biological complexity, while not solely restricted to biochemical and histological readout parameters
The Evolving Druggability and Developability Space Part One: Druggability Part Two: Developability
No full textPart one: This review provides a holistic introduction to the evolving druggability and developability space. Before the genomic era when traditional druggable targets were the main stream of drug discovery, Lipinski’s rule-of-five (Ro5) evolved and served as a guide toward drug-like molecule design for oral delivery. In contrast, during the post-genomic era, a transition has occurred in discovery towards applying new chemistry to bind and modulate challenging biological targets for future innovative treatments. Consequently, drugging the yet-to-be drugged targets using various molecular modalities and discovery strategies is a key focus for drug discovery and development. Herein some of the changes arising during the discovery ‘druggability’ phase are described as well as their potential implications on overall developability. Part 1 discusses the general concepts of druggability and developability, the changes in target identification methods, the shifts in chemical space for new biological targets and finally the anticipated challenges arising from these transitions.
Part Two: Herewithin is the second part of a two part publication on druggability and developability. This article provides a high-level evaluation of the current risks associated with physiochemical properties, ADME properties and formulation approaches for three classes of molecular modalities: complex small molecules, peptides/peptidomimetics, and oligonucleotides. An analysis of the strategies used to progress the most advanced candidate for each category is discussed. Finally, an outlook is provided detailing the trending chemistry and formulation strategies observed across these molecular modalities, which are directly related to their challenging physical-chemical properties