Improving Assessment of Drug Safety Through Proteomics: Early Detection and Mechanistic Characterization of the Unforeseen Harmful Effects of Torcetrapib.

BackgroundEarly detection of adverse effects of novel therapies and understanding of their mechanisms could improve the safety and efficiency of drug development. We have retrospectively applied large-scale proteomics to blood samples from ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), a trial of torcetrapib (a cholesterol ester transfer protein inhibitor), that involved 15 067 participants at high cardiovascular risk. ILLUMINATE was terminated at a median of 550 days because of significant absolute increases of 1.2% in cardiovascular events and 0.4% in mortality with torcetrapib. The aims of our analysis were to determine whether a proteomic analysis might reveal biological mechanisms responsible for these harmful effects and whether harmful effects of torcetrapib could have been detected early in the ILLUMINATE trial with proteomics.MethodsA nested case-control analysis of paired plasma samples at baseline and at 3 months was performed in 249 participants assigned to torcetrapib plus atorvastatin and 223 participants assigned to atorvastatin only. Within each treatment arm, cases with events were matched to controls 1:1. Main outcomes were a survey of 1129 proteins for discovery of biological pathways altered by torcetrapib and a 9-protein risk score validated to predict myocardial infarction, stroke, heart failure, or death.ResultsPlasma concentrations of 200 proteins changed significantly with torcetrapib. Their pathway analysis revealed unexpected and widespread changes in immune and inflammatory functions, as well as changes in endocrine systems, including in aldosterone function and glycemic control. At baseline, 9-protein risk scores were similar in the 2 treatment arms and higher in participants with subsequent events. At 3 months, the absolute 9-protein derived risk increased in the torcetrapib plus atorvastatin arm compared with the atorvastatin-only arm by 1.08% (P=0.0004). Thirty-seven proteins changed in the direction of increased risk of 49 proteins previously associated with cardiovascular and mortality risk.ConclusionsHeretofore unknown effects of torcetrapib were revealed in immune and inflammatory functions. A protein-based risk score predicted harm from torcetrapib within just 3 months. A protein-based risk assessment embedded within a large proteomic survey may prove to be useful in the evaluation of therapies to prevent harm to patients.Clinical trial registrationURL: https://www.clinicaltrials.gov. Unique identifier: NCT00134264

Polycontinuous geometries for inverse lipid phases with more than two aqueous network domains

Inverse bicontinuous cubic phases with two aqueous network domains separated by a smooth bilayer are firmly established as equilibrium phases in lipid/water systems. The purpose of this article is to highlight the generalisations of these bicontinuous geometries to polycontinuous geometries, which could be realised as lipid mesophases with three or more network-like aqueous domains separated by a branched bilayer. An analysis of structural homogeneity in terms of bilayer width variations reveals that ordered polycontinuous geometries are likely candidates for lipid mesophase structures, with similar chain packing characteristics to the inverse micellar phases (that once were believed not to exist due to high packing frustration). The average molecular shape required by global geometry to form these multi-network phases is quantified by the surfactant shape parameter, v/(al); we find that it adopts values close to those of the known lipid phases. We specifically analyse the 3etc(187 193) structure of hexagonal symmetry P63 /mcm with three aqueous domains, the 3dia(24 220) structure of cubic symmetry I 3d composed of three distorted diamond networks, the cubic chiral 4srs(24 208) with cubic symmetry P4232 and the achiral 4srs(5 133) structure of symmetry P42/nbc, each consisting of four intergrown undistorted copies of the srs net (the same net as in the QGII gyroid phase). Structural homogeneity is analysed by a medial surface approach assuming that the head-group interfaces are constant mean curvature surfaces. To facilitate future experimental identification, we provide simulated SAXS scattering patterns that, for the 4srs(24 208) and 3dia(24 220) structures, bear remarkable similarity to those of bicontinuous QGII-gyroid and QDII-diamond phases, with comparable lattice parameters and only a single peak that cannot be indexed to the well-established structures. While polycontinuous lipid phases have, to date, not been reported, the likelihood of their formation is further indicated by the reported observation of a solid tricontinuous mesoporous silicate structure, termed IBN-9, which formed in the presence of surfactants [Han et al., Nat. Chem., 2009, 1, 123]

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression

Assessment of F/HN-Pseudotyped Lentivirus as a Clinically Relevant Vector for Lung Gene Therapy

RATIONALE: Ongoing efforts to improve pulmonary gene transfer thereby enabling gene therapy for the treatment of lung diseases, such as cystic fibrosis (CF), has led to the assessment of a lentiviral vector (simian immunodeficiency virus [SIV]) pseudotyped with the Sendai virus envelope proteins F and HN. OBJECTIVES: To place this vector onto a translational pathway to the clinic by addressing some key milestones that have to be achieved. METHODS: F/HN-SIV transduction efficiency, duration of expression, and toxicity were assessed in mice. In addition, F/HN-SIV was assessed in differentiated human air-liquid interface cultures, primary human nasal epithelial cells, and human and sheep lung slices. MEASUREMENTS AND MAIN RESULTS: A single dose produces lung expression for the lifetime of the mouse (~2 yr). Only brief contact time is needed to achieve transduction. Repeated daily administration leads to a dose-related increase in gene expression. Repeated monthly administration to mouse lower airways is feasible without loss of gene expression. There is no evidence of chronic toxicity during a 2-year study period. F/HN-SIV leads to persistent gene expression in human differentiated airway cultures and human lung slices and transduces freshly obtained primary human airway epithelial cells. CONCLUSIONS: The data support F/HN-pseudotyped SIV as a promising vector for pulmonary gene therapy for several diseases including CF. We are now undertaking the necessary refinements to progress this vector into clinical trials

Real-time analysis of the binding of fluorescent VEGF₁₆₅a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF₁₆₅a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF₁₆₅a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF₁₆₅a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane

CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression.

Pulmonary delivery of plasmid DNA (pDNA)/cationic liposome complexes is associated with an acute unmethylated CG dinucleotide (CpG)-mediated inflammatory response and brief duration of transgene expression. We demonstrate that retention of even a single CpG in pDNA is sufficient to elicit an inflammatory response, whereas CpG-free pDNA vectors do not. Using a CpG-free pDNA expression vector, we achieved sustained (≥56 d) in vivo transgene expression in the absence of lung inflammation

Risk factors for genital infections in people initiating SGLT2 inhibitors and their impact on discontinuation

Introduction: To identify risk factors, absolute risk, and impact on treatment discontinuation of genital infections with sodium-glucose co-transporter-2 inhibitors (SGLT2i). Research design and methods: We assessed the relationship between baseline characteristics and genital infection in 21 004 people with type 2 diabetes initiating SGLT2i and 55 471 controls initiating dipeptidyl peptidase-4 inhibitors (DPP4i) in a UK primary care database. We assessed absolute risk of infection in those with key risk factors and the association between early genital infection and treatment discontinuation. Results: Genital infection was substantially more common in those treated with SGLT2i (8.1% within 1 year) than DPP4i (1.8%). Key predictors of infection with SGLT2i were female sex (HR 3.64; 95% CI 3.23 to 4.11) and history of genital infection; <1 year before initiation (HR 4.38; 3.73 to 5.13), 1–5 years (HR 3.04; 2.64 to 3.51), and >5 years (HR 1.79; 1.55 to 2.07). Baseline HbA1c was not associated with infection risk for SGLT2i, in contrast to DPP4i where risk increased with higher HbA1c. One-year absolute risk of genital infection with SGLT2i was highest for those with a history of prior infection (females 23.7%, males 12.1%), compared with those without (females 10.8%, males 2.7%). Early genital infection was associated with a similar discontinuation risk for SGLT2i (HR 1.48; 1.21–1.80) and DPP4i (HR 1.58; 1.21–2.07). Conclusions: Female sex and history of prior infection are simple features that can identify subgroups at greatly increased risk of genital infections with SGLT2i therapy. These data can be used to risk-stratify patients. High HbA1c is not a risk factor for genital infections with SGLT2i

The Seventh Data Release of the Sloan Digital Sky Survey

This paper describes the Seventh Data Release of the Sloan Digital Sky Survey (SDSS), marking the completion of the original goals of the SDSS and the end of the phase known as SDSS-II. It includes 11663 deg^2 of imaging data, with most of the roughly 2000 deg^2 increment over the previous data release lying in regions of low Galactic latitude. The catalog contains five-band photometry for 357 million distinct objects. The survey also includes repeat photometry over 250 deg^2 along the Celestial Equator in the Southern Galactic Cap. A coaddition of these data goes roughly two magnitudes fainter than the main survey. The spectroscopy is now complete over a contiguous area of 7500 deg^2 in the Northern Galactic Cap, closing the gap that was present in previous data releases. There are over 1.6 million spectra in total, including 930,000 galaxies, 120,000 quasars, and 460,000 stars. The data release includes improved stellar photometry at low Galactic latitude. The astrometry has all been recalibrated with the second version of the USNO CCD Astrograph Catalog (UCAC-2), reducing the rms statistical errors at the bright end to 45 milli-arcseconds per coordinate. A systematic error in bright galaxy photometr is less severe than previously reported for the majority of galaxies. Finally, we describe a series of improvements to the spectroscopic reductions, including better flat-fielding and improved wavelength calibration at the blue end, better processing of objects with extremely strong narrow emission lines, and an improved determination of stellar metallicities. (Abridged)Comment: 20 pages, 10 embedded figures. Accepted to ApJS after minor correction