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PET Imaging of T Cells: Target Identification and Feasibility Assessment
No full textImaging Tcells using positron emission tomography (PET) would be highly useful for diagnosis and monitoring in immunology and oncology patients. There are, however, no obvious targets that can be used to develop imaging agents for this purpose. We evaluated several potential target proteins with selective expression in Tcells, and for which lead molecules were available: protein kinaseC isozymeθ (PKCθ), lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase70 (ZAP70), and interleukin-2-inducible T-cell kinase (Itk). Ultimately, we focused on Itk and identified a tool molecule with properties suitable for in vivo imaging of Tcells: (5aR)-5,5-difluoro-5a-methyl-N-(1-((S)-3-(methylsulfonyl)phenyl)(tetrahydro-2H-pyran-4-yl)methyl)-1H-pyrazol-4-yl)-1,4,4a,5,5a,6-hexahydrocyclopropa[f]indazole-3-carboxamide (23). Although it does not have the optimal profile for clinical use, this molecule indicates that it might be possible to develop Itk-selective PET ligands for imaging the distribution of Tcells in patients
Phase 1/1b dose escalation and expansion study of BEZ235, a dual PI3K/mTOR inhibitor, in patients with advanced solid tumors including patients with advanced breast cancer
No full textPurpose: To determine the maximum tolerated dose (MTD) of BEZ235, an oral inhibitor of class I PI3K and mTOR complexes 1 and 2. Methods: We performed a phase I/Ib, multicenter, open-label study of oral BEZ235 administered in a continuous daily schedule. The study consisted of two parts: dose-escalation part and safety-expansion part. BEZ235 was administered as a single agent to patients with solid tumors or in combination with trastuzumab for HER2+ advanced breast cancer (aBC). Primary end points were MTD, safety, and tolerability. The secondary end point was pharmacokinetics. Other formulations of BEZ235, solid dispersion system (SDS) sachet, and SDS capsules were also assessed. Results: One hundred and eighty-three patients were enrolled; single-agent BEZ235 was administered as hard gelatin capsule (n = 59), SDS capsules A and B (n = 33), and SDS sachet (n = 61), amongst which SDS sachet was chosen as the preferred formulation. The monotherapy MTD for capsule A and SDS sachet was determined to be 1000 and 1200 mg/day, respectively. Thirty patients with HER2+ aBC received BEZ235 in combination with trastuzumab. The MTD of BEZ235 in combination with trastuzumab was 600 mg/day. A total of four patients (13.3%) achieved partial response across the different groups. Most frequent AEs in single agent and combination cohorts included nausea (80.3 and 93.3%), diarrhea (75.4 and 80.0%), and vomiting (63.9 and 63.3%). Conclusions: The MTD of BEZ235 as single agent was 1200 and 600 mg/day with trastuzumab. Pharmacokinetic profiles showed low-to-moderate variability at low dose (10 mg) and high variability at high doses (100 mg and above). Gastrointestinal AEs were frequent at high doses
Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out
No full textAn increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects.
Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using “Transcription Activator-Like Effector Nucleases” (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins.
The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in conditioned medium derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line.
In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins.
Optimization of 3-Pyrimidin-4-yl-oxazolidin-2-ones as Orally Bioavailable and Brain Penetrant Mutant IDH1 Inhibitors
No full textMutant isocitrate dehydrogenase 1 (IDH1) is an attractive therapeutic target for the treatment of various cancers such as AML, glioma and glioblastoma. We have evaluated 3-pyrimidin-4-yl-oxazolidin-2-ones as mutant IDH1 inhibitors that bind to an allosteric, induced pocket of IDH1R132H. This letter describes SAR exploration focused on improving both the in vitro and in vivo metabolic stability of the compounds, leading to the identification of 19 as a potent and selective mutant IDH1 inhibitor that has demonstrated brain penetration and excellent oral bioavailability in rodents. In a preclinical patient-derived IDH1 mutant xenograft tumor model study, 19 efficiently inhibited the production of the biomarker, 2-HG
Scalable Microfluidic Platform for Flexible Configuration of and Experiments with Microtissue Multiorgan Models
No full textMicrophysiological systems (MPS) are a promising solution, to bridge current gaps in the predictive power of existing techniques in industrial substance testing (i.e., pharmaceutical, chemical, cosmetic and tobacco industries), as they mimic human physiology in vitro. A variety of microfluidics-based MPS have recently been developed. They can be split into three groups: (i) single-organ or “organ-on-a-chip”, (ii) multi-organ, and (iii) “body-on-a-chip” systems.1 “Organ-on-a-chip” systems focus mainly on a realistic representation of an organ’s multicellular microarchitecture and functionality. Multi-organ systems with two or more organs focus on specific organ interaction and interplay
INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling
No full textThe inflammatory bowel disease risk-gene INAVA is associated with regulation of
epithelial barrier integrity and innate immune signaling. Here, we define the molecular basis for
INAVA in epithelial F-actin assembly and inflammation. INAVA acts through its DUF3338
domain (renamed CUPID) to bind and recruit ARNO, a cytohesin ARF-GEF member, to lateral
membranes and enhance cortical F-actin assembly. Strikingly, this process is GEF activity
independent, implicating a novel and non-canonical mechanism for cytohesins. We find a second
function for INAVA in enhancing polyubiquitination in innate immune signaling cascades
downstream of IL-1β and NOD2. In this case, ARNO binding inhibits CUPID activity to
negatively-regulate inflammatory responses. Thus, INAVA exhibits dual functions coordinated
directly by ARNO that bridge epithelial barriers with inflammatory signaling at mucosal surfaces
Enabling drug discovery and development through single-cell imaging
No full textSingle-cell imaging-based assays are an area of active and growing investment in drug discovery and development. This approach offers researchers the capability to interrogate rare subpopulations of cells with minimal sample consumption and multiplexed readouts. Recent technological advances in the optical interrogation and manipulation of single cells have substantially increased the throughput and sensitivity of these assays. Areas covered: In this review, the authors focus on three classes of single-cell imaging-based analyses: single-cell microscopy combined with microfluidics, mass spectrometric imaging for subcellular compound localization, and imaging mass cytometry (IMC). They provide an overview of each technology and recent examples of their utility in advancing drug discovery, based on the potential for scalability, multiplexing, and capability to generate definitive data on cellular heterogeneity and target engagement. Expert opinion: Understanding target engagement and heterogeneity at the single-cell level will enable the development of safer and more effective therapies, particularly for new modalities like CAR-T cell therapies and gene editing approaches (AAV, CRISPR). Successful adoption of new single-cell imaging-based approaches in drug discovery will require tandem investment in advanced computational analysis and bioinformatic approaches, due to the complexity and multivariate nature of single-cell imaging data
Towards frailty biomarkers: Candidates from genes and pathways regulated in aging and age-related diseases
No full textObjective: Use of the frailty index to measure an accumulation of deficits has been proven a valuable method for identifying elderly people at risk for increased vulnerability, disease, injury, and mortality. However, complementary molecular frailty biomarkers or ideally biomarker panels have not yet been identified. We conducted a systematic search to identify biomarker candidates for a frailty biomarker panel. Methods: Gene expression databases were searched (http://genomics.senescence.info/genes including GenAge, AnAge, LongevityMap, CellAge, DrugAge, Digital Aging Atlas) to identify genes regulated in aging, longevity, and age-related diseases with a focus on secreted factors or molecules detectable in body fluids as potential frailty biomarkers. Factors broadly expressed, related to several “hallmark of aging” pathways as well as used or predicted as biomarkers in other disease settings, particularly age-related pathologies, were identified. This set of biomarkers was further expanded according to the expertise and experience of the authors. In the next step, biomarkers were assigned to six “hallmark of aging” pathways, namely (1) inflammation, (2) mitochondria and apoptosis, (3) calcium homeostasis, (4) fibrosis, (5) NMJ (neuromuscular junction) and neurons, (6) cytoskeleton and hormones, or (7) other principles and an extensive literature search was performed for each candidate to explore their potential and priority as frailty biomarkers. Results: A total of 44 markers were evaluated in the seven categories listed above, and 19 were awarded a high priority score, 22 identified as medium priority and three were low priority. In each category high and medium priority markers were identified. Conclusion: Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFβ (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential
Oxford DTC proposals for 2019 brouchure
No full textTwo proposals to submit to the Oxford 2019 DTC brochure as part of Novartis sponsorship of the program:
1) Proposal 1: Innovative Synthesis of Small Ring Chiral Building Blocks
2) Proposal 2: Diversity Driven sp2-sp3 cross couplings for plate enabled Microscale Synthesis
Proposals contain no Novartis IP, and cite only literature examples
Zoledronic Acid Increases the Prevalence of Medication-related Osteonecrosis of the Jaw in Rice Rats (Oryzomys palustris) in a Dose Dependent Manner
No full textOBJECTIVE: Investigate role of dose/duration of zoledronic acid (ZOL), a powerful anti-resorptive (pAR), on prevalence of medication-related osteonecrosis of the jaw (MRONJ) in rice rats (Oryzomys palustris), a species with natural susceptibility to food-impaction induced localized periodontitis (FILP) when fed standard (STD) rodent chow. We hypothesize that ZOL dose/duration predicts MRONJ prevalence.
METHODS: We performed a toxicology experiment with clinically-relevant doses of ZOL in female rats (N=230) fed STD rodent chow. At weaning (age 4 wks; baseline), 12 rats were necropsied. The rest were randomized into five groups that immediately began to receive 0, 8, 20, 50 or 125µg/kg ZOL IV/q 4 wks. After 12, 18, 24 and 30 wks, rat groups from each dose (N=9-16) were necropsied. High-resolution, macroscopic photos of all jaw quadrants were given a gross quadrant grade (GQG, 0-4) that classified lesion severity and determined presence of gross MRONJ lesions. Quadrants with GQG≥1 were examined histopathologically. Multiple logistic regression analysis (ZOL dose/time) of ONJ prevalence was completed. RESULTS: We found: 1) baseline rats and rats treated with 0µg/kg ZOL had no MRONJ; 2) 75% of 0µg/kg ZOL rats developed FILP lesions; 3) by gross observation, 29 MRONJ cases were identified; 4) by histopathology, all gross MRONJ cases were confirmed and 53 new cases were discovered; 5) ZOL dose (p = ~0.001), but not duration (p = ~0.32), was a significant predictor of MRONJ prevalence; 6) 13% prevalence of gross MRONJ, with 26% prevalence among rats exposed to ZOL oncology doses; 7) 36% prevalence of histopathological MRONJ, with 73% prevalence among rats exposed to ZOL oncology doses. CONCLUSIONS: This animal study demonstrates a very high MRONJ prevalence with a positive relationship of relevant ZOL dose to MRONJ prevalence