F. Hoffmann LaRoche Ltd.
F. Hoffmann LaRoche Ltd.
Kennedy E.M.,Duke University |
Bassit L.C.,Emory University |
Mueller H.,F. Hoffmann LaRoche Inc. |
Kornepati A.V.R.,Duke University |
And 6 more authors.
Virology | Year: 2015
Hepatitis B virus (HBV) remains a major human pathogen, with over 240 million individuals suffering from chronic HBV infections. These can persist for decades due to the lack of therapies that can effectively target the stable viral covalently closed circular (ccc) DNA molecules present in infected hepatocytes. Using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs (sgRNAs) specific for HBV, we observed effective inhibition of HBV DNA production in in vitro models of both chronic and de novo HBV infection. Cas9/sgRNA combinations specific for HBV reduced total viral DNA levels by up to ~1000-fold and HBV cccDNA levels by up to ~10-fold and also mutationally inactivated the majority of the residual viral DNA. Together, these data provide proof of principle for the hypothesis that CRISPR/Cas systems have the potential to serve as effective tools for the depletion of the cccDNA pool in chronically HBV infected individuals. © 2014 Elsevier Inc.
Wallace T.L.,Hoffmann-La Roche |
Callahan P.M.,Memory Pharmaceuticals |
Tehim A.,Memory Pharmaceuticals |
Bertrand D.,HiQ Screening Sarl |
And 13 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2011
Neuronal nicotinic α7 acetylcholine receptors (α7nAChRs) are expressed primarily in the brain and are implicated in modulating many cognitive functions (e.g., attention, working and episodic memory). Not surprisingly, much effort has been committed to the development of molecules acting at α7nAChRs as potential therapies for a variety of central nervous system diseases (e.g., Alzheimer's). N-[(3S)-1-azabicyclo[2.2.2]oct-3-yl]-1H-indazole- 3-carboxamide hydrochloride (RG3487) binds potently to the human α7nAChR (Ki = 6 nM), in which it acts as a partial agonist (63-69% of acetylcholine) as assessed by whole-cell patch-clamp recordings in both oocytes and QM7 cell lines. RG3487 activates human α7nAChRs with an EC 50 of 0.8 μM (oocytes) and 7.7 μM (QM7 cells). RG3487 also exhibits antagonist properties at the serotonin 3 receptor [IC50 = 2.8 nM (oocytes), 32.7 nM (N1E-115 cells)]. In vivo, RG3487 improved object recognition memory in rats after acute [minimally effective dose (MED) 1.0 mg/kg p.o.] or repeated (10 day) administration at brain and plasma concentrations in the low-nanomolar range. Spatial learning deficits in age-impaired rats were reversed after RG3487 administration (MED: 0.03 mg/kg i.p.) as evaluated in the Morris water maze task. In the prepulse inhibition (PPI) of startle model of sensorimotor gating, RG3487 improved apomorphine-induced deficits in PPI performance (MED: 0.03 mg/kg i.p.) and reversed phencyclidine-induced impairments in an attentional set-shifting model of executive function (MED: ≤0.03 mg/kg i.p.). Cumulative evidence from these studies indicates RG3487 is a novel and potent α7nAChR partial agonist that improves cognitive performance and sensorimotor gating. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.
Scharfer C.,University of Hamburg |
Schulz-Gasch T.,F.Hoffmann LaRoche Ltd. |
Hert J.,F.Hoffmann LaRoche Ltd. |
Heinzerling L.,University of Hamburg |
And 4 more authors.
ChemMedChem | Year: 2013
The generation of sets of low-energy conformations for a given molecule is a central task in drug design. Herein we present a new conformation generator called CONFECT that builds on our previously published library of torsion patterns. Conformations are generated as well as ranked by means of normalized frequency distributions derived from the Cambridge Structural Database (CSD). Following an incremental construction approach, conformations are selected from a systematic enumeration within energetic boundaries. The new tool is benchmarked in several different ways, indicating that it allows the efficient generation of high-quality conformation ensembles. These ensembles are smaller than those produced by state-of-the-art tools, yet they effectively cover conformational space. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Quebatte G.,University of Basel |
Kitas E.,F. Hoffmann LaRoche Ltd. |
Seelig J.,University of Basel
Journal of Physical Chemistry B | Year: 2013
DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 107 M-1 and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 106 M-1 and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 106 M-1. Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin. © 2013 American Chemical Society.
Paynter N.P.,Brigham and Women's Hospital |
Mazer N.A.,F. Hoffmann LaRoche Ltd. |
Pradhan A.D.,Brigham and Women's Hospital |
Gaziano J.M.,Brigham and Women's Hospital |
And 3 more authors.
Archives of Internal Medicine | Year: 2011
Background: It is unclear whether models that include hemoglobin A 1c (HbA 1c) levels only for diabetic patients improve the ability to predict cardiovascular disease (CVD) risk compared with the currently recommended classification of diabetes as a cardiovascular risk equivalent. Methods: A total of 24 674 women (including 685 diabetic participants at baseline) and 11 280 men (including 563 diabetic participants at baseline) were followed up prospectively for cardiovascular disease (CVD). One hundred twenty-five CVD events occurred in diabetic women (666 in nondiabetic women), and 170 events occurred in diabetic men (1382 in nondiabetic men). Models for CVD risk were generated separately for men and women using the traditional CVD risk factors with the addition of a term for HbA 1c levels only for diabetic individuals. In diabetic participants, the resulting predicted risks were compared with classification of diabetes as a cardiovascular risk equivalent (10-year CVD risk of at least 20%). Results: In women, the models including HbA 1c levels in diabetic participants improved the C statistic by 0.177 (P<.001) over the risk equivalence model and showed improved reclassification (net reclassification improvement [NRI] of 26.7% [P=.001]). In men, the improvements were more modest but still statistically significant (C statistic change of 0.039 [P=.02]; NRI of 9.2% [P=.04]). Including HbA 1c levels also improved prediction over a dichotomous term for diabetes in women (NRI of 11.8% [P=.03]) but not in men. Conclusions: In both women and men with diabetes at baseline, we observed significant improvements in predictive ability of CVD risk using models incorporating HbA 1c levels compared with classification of diabetes as a cardiovascular risk equivalent. ©2011 American Medical Association. All rights reserved.
Zimmermann H.,University of Kiel |
Reinke P.,Charité - Medical University of Berlin |
Neuhaus R.,Charité - Medical University of Berlin |
Lehmkuhl H.,German Heart Institute Berlin |
And 9 more authors.
Cancer | Year: 2012
BACKGROUND: Burkitt lymphoma post-Transplantation lymphoproliferative disorder (Burkitt-PTLD) is a rare form of monomorphic B-cell PTLD for which no standard treatment has been established. Currently, the treatment of Burkitt lymphoma outside the post-Transplantation setting involves high doses of alkylating agents, frequent dosing, and intrathecal and/or systemic central nervous system prophylaxis. In PTLD, however, such protocols are associated with considerable toxicity and mortality. METHODS: The authors present a retrospective series of 8 adult patients with Burkitt-PTLD. Six patients were reported to the prospective German PTLD registry or were enrolled in the PTLD-1 trial, and 2 patients had received treatment before 2000, thus allowing for comparison with the pre-rituximab era. RESULTS: Seven of the 8 patients were men. The median age at presentation was 38 years, and the median time since transplantation was 5.7 years. Five of 8 patients had histologically established, Epstein-Barr virus-associated disease, and 7 of 7 patients were positive for a MYC translocation. Five of 8 patients received sequential immunochemotherapy (4 courses of rituximab [R] followed by 4 cycles of cyclophosphamide, doxorubicin, vincristine, and prednisolone [CHOP] or R plus CHOP [R-CHOP]). In this group, 5 of 5 patients reached complete remission (CR), and their overall survival (OS) was significantly longer (P =.008) compared with the OS for 2 of 8 patients who received first-line CHOP and did not respond. One of 8 patients (who had stage IV disease with meningiosis) received combination therapy (cyclophosphamide pretreatment, rituximab, intrathecal chemotherapy, whole-brain irradiation, and radioimmunotherapy) and reached CR. Overall, 6 of 8 patients reached CR; and, after a median follow-up of 4.7 years (range, 1.7-4.8 years), the median OS was 36.7 months. There was no treatment-related mortality under first-line therapy. CONCLUSIONS: In the largest adult case series in Burkitt-PTLD to date, sequential immunochemotherapy with rituximab followed by standard CHOP or R-CHOP was a both safe and effective treatment. © 2012 American Cancer Society.
Pavlidis I.V.,University of Greifswald |
Pavlidis I.V.,University of Kassel |
Weiss M.S.,University of Greifswald |
Genz M.,University of Greifswald |
And 5 more authors.
Nature Chemistry | Year: 2016
The use of transaminases to access pharmaceutically relevant chiral amines is an attractive alternative to transition-metal-catalysed asymmetric chemical synthesis. However, one major challenge is their limited substrate scope. Here we report the creation of highly active and stereoselective transaminases starting from fold class I. The transaminases were developed by extensive protein engineering followed by optimization of the identified motif. The resulting enzymes exhibited up to 8,900-fold higher activity than the starting scaffold and are highly stereoselective (up to >99.9% enantiomeric excess) in the asymmetric synthesis of a set of chiral amines bearing bulky substituents. These enzymes should therefore be suitable for use in the synthesis of a wide array of potential intermediates for pharmaceuticals. We also show that the motif can be engineered into other protein scaffolds with sequence identities as low as 70%, and as such should have a broad impact in the field of biocatalytic synthesis and enzyme engineering. © 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
Schnueriger A.,F. Hoffmann LaRoche Ltd |
Grau R.,F. Hoffmann LaRoche Ltd |
Sondermann P.,F. Hoffmann LaRoche Ltd |
Schreitmueller T.,F. Hoffmann LaRoche Ltd |
And 2 more authors.
Molecular Immunology | Year: 2011
Antibody-dependent cellular cytotoxicity (ADCC) contributes to clinical efficacy of a broad range of antibody therapeutics. However, reproducible quantitation of ADCC activity on a cellular level remains highly challenging, as ADCC assays rely on primary effector cells associated with laborious cell purification procedures, resulting in highly donor-dependent results. Here, we report the development of an in vitro ADCC method based on an engineered human natural killer cell line as effectors. While eliminating the limitations of primary cells, this assay exhibits all the hallmarks of traditional ADCC assay systems. We have used this assay to measure the ADCC activity of a humanized IgG1 antibody directed against the human CD20 antigen. Our data show that this assay is capable to measure small changes in ADCC and can therefore be used to test therapeutic antibodies against cell-surface targets for their depleting activity. © 2011 Elsevier Ltd.
Venet D.,International Drug Development Institute IDDI |
Venet D.,Free University of Brussels |
Doffagne E.,International Drug Development Institute IDDI |
Burzykowski T.,International Drug Development Institute IDDI |
And 10 more authors.
Clinical Trials | Year: 2012
Background Classical monitoring approaches rely on extensive on-site visits and source data verification. These activities are associated with high cost and a limited contribution to data quality. Central statistical monitoring is of particular interest to address these shortcomings. Purpose This article outlines the principles of central statistical monitoring and the challenges of implementing it in actual trials. Methods A statistical approach to central monitoring is based on a large number of statistical tests performed on all variables collected in the database, in order to identify centers that differ from the others. The tests generate a high-dimensional matrix of p -values, which can be analyzed by statistical methods and bioinformatics tools to identify extreme centers. Results Results from actual trials are provided to illustrate typical findings that can be expected from a central statistical monitoring approach, which detects abnormal patterns that were not (or could not have been) detected by on-site monitoring. Limitations Central statistical monitoring can only address problems present in the data. Important aspects of trial conduct such as a lack of informed consent documentation, for instance, require other approaches. The results provided here are empirical examples from a limited number of studies. Conclusion Central statistical monitoring can both optimize on-site monitoring and improve data quality and as such provides a cost-effective way of meeting regulatory requirements for clinical trials. © The Author(s), 2012.
PubMed | e Oncology Information Service, c SVA Zurich, University of Cologne, d PIOH and F. Hoffmann LaRoche Ltd.
Type: Journal Article | Journal: Leukemia & lymphoma | Year: 2016
The cost-effectiveness of rituximab in combination with fludarabine/cyclophosphamide (R-FC) for the first line treatment of chronic lymphocytic leukemia (CLL) was evaluated. Based on long-term clinical data (follow-up of 5.9 years) from the CLL8-trial, a Markov-model with three health states (Free from disease progression, Progressive disease, Death) was used to evaluate the cost per quality-adjusted life-year (QALY) and cost per life years gained (LYG) of R-FC from the perspective of the German statutory health insurance (SHI). The addition of rituximab to FC chemotherapy results in a gain of 1.1 quality-adjusted life-years. The incremental cost-effectiveness ratio (ICER) of R-FC compared with FC was 17,979 per QALY (15,773 per LYG). Results were robust in deterministic and probabilistic sensitivity analyses. From the German SHI perspective, rituximab in combination with FC chemotherapy represents good value for first-line treatment of patients with CLL and compares favorably with chemotherapy alone.