7196 research outputs found
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Understanding Sjögren’s disease at the single cell level
No full textThis a piece of text to gain access to publicly available restricted human dataset. This will appear on an NIIH website with the Novartis company name if approved
Modulating antibody N-glycosylation through feed additives using a multi-tiered approach
No full textGlycosylation of recombinant proteins is a post-translational modification that affects multiple physicochemical and biological properties of proteins. As such, it is a critical quality attribute that must be carefully controlled during protein production in the pharmaceutical industry. Glycosylation can be modulated by various conditions, including the composition of production media and feeds. In this study, the N-glycosylation-modulating effects of numerous compounds, including metal enzyme cofactors, enzyme inhibitors, and metabolic intermediates, were evaluated. Chinese hamster ovary cells producing three different IgG antibodies were cultivated in a fed-batch mode. First, a one-factor-at-a-time experiment was performed in 24-well deep well plates to identify the strongest modulators and appropriate concentration ranges. Then, a full response surface experiment was designed to gauge the effects and interactions of the 14 most effective hit compounds in an Ambr® 15 bioreactor system. A wide range of glycoform content was achieved, with an up to eight-fold increase in individual glycoforms compared to controls. The resulting model can be used to determine modulator combinations that will yield desired glycoforms in the final product
Data sharing for the Cross-Pharma Complex Nitrosamines Collaborative Project
No full textResults of the enhanced Ames test for 4-Methoxy-N-[(4-Methoxy-phenyl)Methy]-N-Nitroso-Benzenemethanamamine, CAS 19873-43-9 will be shared with Cross-Pharma Complex Nitrosamines Collaborative Project
Signed project terms are attached.
The structure of the nitrosamine is obvious in public domain, it has a CAS number.
It is related to TWN172
Data sharing for the Cross-Pharma Complex Nitrosamines Collaborative Project
No full textResults of the Ames test (non-GLP) for 6'-fluoro-1'-nitroso-1'H-spiro[piperidine-4,2'-quinolin]-4'(3'H)-one will be shared with Cross-Pharma Complex Nitrosamines Collaborative Project Signed project terms are attached. The nitrosamine is related to MGY825
A spontaneous mutation in ADIPOR1 causes retinal degeneration in mice
No full textAdiponectin receptor 1 (ADIPOR1) is a transmembrane protein necessary for normal anatomy and physiology in the retina. In a recent study of complement factor H knockout mice (Cfh-/-), our lab discovered a flecked retina phenotype and retinal thinning by fundus imaging and optical coherence tomography (OCT), respectively. The phenotype was observed in a subset (50%) of Cfh-/- mice. The thinning observed in vivo is due to an early degeneration of rod photoreceptors. This phenotype has not been reported in published studies of Cfh-/- mice. AdipoR1 knockout mice (AdipoR1-/-) and mice deficient in Membrane Frizzled Related Protein (MFRP) exhibit this phenotype, suggesting an involvement in the emergence of the retinal degeneration observed in a subset of Cfh-/- mice. Cfh and AdipoR1 are located in close proximity on mouse Chromosome 1 (Chr1) and a complementation cross between Cfh and AdipoR1 mice with retinal degeneration produced 100% progeny with retinal degeneration. Sequencing of the Cfh-/- mice revealed a c.841 C > T mutation in AdipoR1. Furthermore, one Cfh wildtype (of Cfh+/+) and 2 heterozygous (of Cfh+/-) mice exhibited retinal degeneration and were homozygous for the point mutation. The c.841 C > T mutation results in a proline to serine conversion at position 281 (P281S) in ADIPOR1. This residue is critical for ADIPOR1 open and closed conformations in the membrane. In silico modeling of candidate ADIPOR1 ligands, 11-cis-retinaldehyde and docosahexaenoic acid (DHA), that are deficient in AdipoR1-/-, suggests that ADIPOR1 is involved in trafficking retinoids and fatty acids and their combined deficiency in the ADIPOR1 mutant retinas might explain the retinal degeneration phenotype
Population modeling of nilotinib exposure versus longitudinal BCR::ABL1 response in patients with chronic phase chronic myeloid leukemia using a semi-mechanistic disease model
No full textBackground: This study evaluated the exposure-efficacy relationship of nilotinib and longitudinal BCR::ABL1 levels in patients with newly diagnosed Philadelphia chromosome–positive chronic myeloid leukemia in chronic phase (Ph+ CML-CP) and those who are imatinib-resistant or intolerant using a semi-mechanistic model.
Methods: The analysis included 489 CML-CP patients from three nilotinib trials (NCT00109707; NCT00471497; NCT01043874) with duration of follow-up ranging from 2 to 9 years. The semi-mechanistic disease model of CML-CP consisted of quiescent leukemic stem cells (q), proliferating drug-susceptible (p) and resistant (r) bone marrow cells. Drug effect on the elimination of p cells was characterized by an Emax model based on the individual daily AUC0-24h simulated using their empirical Bayes estimates from a population pharmacokinetic model. The influence of line of therapy was evaluated on model parameters and its impact was investigated through simulations of the major molecular response (MMR) rate, defined as the proportion of the simulated profiles that achieved BCR::ABL1 level of ≤0.1% at 48 and 96 weeks of treatment.
Results: The final disease model was based on a truncated 3-year data that characterized the bi-phasic pattern of BCR::ABL1 transcript profiles. Line of therapy was a significant covariate of the drug kill effect, p and r cells. Simulations of BCR::ABL1 time course predicted MMR rates at 48 weeks and 96 weeks for both nilotinib 300 and 400 mg twice-daily of 66-71% and 77-82% in first-line, and 34-39% and 46-54% in second-line, respectively. Results are consistent with observed MMR rates in the respective trials.
Conclusions: The current disease model was developed using time-course of BCR::ABL1 transcript profiles of nilotinib in first- and second-line CML-CP. The ability to distinguish molecular response between lines of therapy is demonstrated using model-based analysis. These nilotinib information enable the extrapolation of novel TKI’s (e.g., asciminib) response to other lines of therapy in patients with CML-CP
Adeno-associated virus serotype 2 with proteolytic cut by trypsin remains intact and potent
No full textRecombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity, immunogenicity, and play important roles in receptor binding, viral escape from the endosome, and transportation of viral DNA to the nucleus. In the journey of AAV vector, AAV vectors can be exposed to different proteolytic environments inside the production cells, during the cell lysis step prior to downstream purification, within endosome, and finally inside the cell nucleus. Therefore, evaluation of AAV stability using a proteolytic approach can provide valuable information for engineering AAV vectors for AAV-based gene therapy development. The stability of a modified AAV serotype 2 (AAV2) capsid proteins was evaluated via a proteolytic approach using trypsin and other proteases. Proteolytic digestion of the AAV2 capsid with trypsin results in clips of the capsid proteins at C-terminus as confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and reversed-phase LC-MS. It was found that the AAV2 capsid with clips not only remains structurally intact, as confirmed by native (non-denaturing) methods including SEC, thermos stability testing, and Cryogenic electron microscopy (cryo-EM), and also remains potent, as confirmed in a potency assay. This finding reveals that the icosahedral three-dimensional structural arrangement of AAV capsid proteins can protect the clipped fragment being released from the capsid, such that the AAV capsid remains intact allowing for the functionality to be maintained to deliver the DNA in the host cell. The finding is valuable for engineering AAV capsids to develop AAV-based gene therapy
Rapid-response RNA-FISH assay platform for coronavirus antiviral high-throughput screening
No full textOver the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses. The first of these outbreaks was the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, which occurred from 2002 to 2004 and resulted in over 700 deaths. Following this, the Middle East respiratory syndrome coronavirus (MERS-CoV) epidemic emerged in 2012, causing over 2600 infections with a case-fatality rate of 36%. More recently, the world has become severely impacted by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of the COVID-19 pandemic, responsible for causing nearly 7 million deaths worldwide. It is crucial to note that the threat of further coronavirus outbreaks continues to be a significant concern, as evidenced by the recent identification of the novel canine CoV (CCoV-HuPn-2018) in several patients with pneumonia in Malaysia. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence in situ hybridization (FISH) assay as a high-throughput rapid response platform for antiviral drug discovery. The flexibility of RNA-FISH probe sets allows for design of viral genome specific probes, enabling in vitro assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors quickly. Screening of 170 antiviral compounds in concentration-response demonstrates a strong R2 correlation between the RNA-FISH assay and a gold-standard immunofluorescence assay for both human CoVs OC43 and 229E. Additionally, we successfully applied this methodology in the context of CCoV 1-71, proving rapid development and deployment, thus, opening new avenues for the evaluation of antiviral drugs to future emerging threats
Identification of TAK-756, a potent and selective TAK1 inhibitor for the treatment of osteoarthritis through intra-articular administration
No full textOsteoarthritis (OA) is a chronic and degenerative joint disease affecting more than 500 million patients worldwide. It is characterized by cartilage degeneration along with low grade synovial inflammation. The onset of OA is multifactorial (e.g. ageing, trauma, obesity and/or genetic) but typically converges towards a progressive diminution of patient’s mobility, and eventually, a deterioration in health. The societal burden of OA is not easily quantifiable but is compounded by the fact that no disease-modifying treatments have been approved to date. Patients are prescribed management therapies (e.g. physical activity programs, viscosupplementation and/or pain killers) until surgical interventions become inevitable (last resort option being a total knee replacement procedure). In this context, several publications report on the transforming growth factor -activated kinase 1 (TAK1) as a potential molecular target for inflammatory arthritides, with complementary anti-catabolic and anti-inflammatory effects. However, severe toxicity has been reported for various TAK1 transgenic mice models, constituting an important hurdle in the development of a chronic treatment for OA. We report herein on the development of TAK1 inhibitors with physicochemical properties suitable for intra-articular injection. Indeed, the injection of a local depot often leads to high drug concentration in the targeted joint, while maintaining low systemic exposure, hence improving the overall safety profile. The desired drug candidate would have to provide long-lasting exposure in the knee joint to reduce the number of intra-articular injections and increase patient compliance. Optimization towards this target product profile placed us in an unusual property space and notably required us to optimize for insolubility in simulated biofluids. More specifically, reducing solubility by increasing crystallinity, while maintaining moderate lipophilicity proved to be a good compromise to ensure high and sustained free drug exposures in the joint. Finally, TAK-756 was discovered as a potent and selective TAK1 inhibitor with excellent intra-articular pharmacokinetic properties
Reactions of in situ-generated difluorocarbene (:CF2) with aromatic/heteroaromatic alcohols, thiols, olefins, and alkynes under environmentally responsible conditions
No full textEnvironmentally respectful methods for generating and utilizing difluorocarbene (:CF2) in the synthesis of a wide array of valuable difluoromethylated compounds are disclosed. In particular, insertion of the CF2 moiety into aromatic/heteroaromatic alcohols, thiols, olefins, and alkynes under neat or aqueous micellar catalysis conditions is demonstrated. These methods yield both satisfactory results and significantly lower E-Factors compared to traditional synthetic approaches. Key applications of these methodologies include optimization en route to a pantoprazole intermediate, and development of a representative one-pot chemoenzymatic sequence. Additionally, analysis via calorimetry indicated no significant safety risk in the context of the developed solvent-free conditions