7196 research outputs found
Sort by
Investigation of Silanol Catalyzed Generation of Cyclopenta[c]Pyrrole Derivative Artifact Peaks During RP-HPLC Impurity Profiling
No full textDuring the stability studies of a new chemical entity (NCE-01), it was observed that a couple of impurities were appearing in both standard and stability samples. Initial analysis report indicated that they were absent in both standard and drug substance loaded for stability. The mass spectrometric analysis indicated that both could be oxidative cyclized cyclopenta[c]pyrrole derivative products, which were also observed during the photolytic forced degradation studies. However, their presence was not consistent during stability analysis, and they were behaving like ghost peaks. Subsequent investigations revealed that these degradation products (ghost peaks) were generated during analysis within the column on the loss of two hydrogens, most likely on interaction
of the drug substance with free silanol(s) and/or residual amount of heavy metals. Catalytic effect of metals potentially present in the column such as ruthenium (III), iron (III), nickel (II), chromium (III), and titanium (IV) was evaluated outside the column by exposing the drug substance with them, however, except ruthenium, other heavy metals failed to produce these impurities. To confirm the catalytic role of ruthenium, another drug analog was also tested, and that too showed the formation of similar degradation products. A mitigation strategy was then developed to reduce/avoid the generation of such on-column ghost peaks by implementing measures such as increasing the buffer strength and substituting acetonitrile with methanol in the mobile phase
Application-based calibration method for calorimeter FlexyTSC SEDEX
No full textThe SYSTAG calorimeter FlexyTSC SEDEX is utilized in Novartis safety laboratories to screen the thermal stability of
reaction mixtures in pharmaceutical synthetic processes. In order to improve the accuracy of SEDEX calorimetric tests,
a customized calibration method was developed specifically for the application scenarios of the calorimeter within our
laboratories. During the check test of the new calibration method, it is no longer to detect the endothermic enthalpies of
the melting process of reference solids, but to detect a known electrical power input through a heating resistor inserted into
the silicone oil within the autoclave (sample cell) under continuous stirring. The check test conditions closely mirror those
of the actual tests performed on chemical reaction mixtures. The new calibration method not only significantly improves
the accuracy of the SEDEX test for detecting exothermic heat but also enhances the efficiency of the calibration process.
It provides a valuable model for optimizing existing calibration schemes or developing new ones for calorimeters in safety
laboratories based on their practical applications. In developing this calibration method, the reaction calorimeter (RC1) was
used to validate the electrical energy output of the heating resistor, while the differential scanning calorimeter (DSC) was
employed for cross-validation after the calibration of SEDEX. This endeavor exemplifies how we can make full use of the
advantages of various calorimetric technologies in a safety laboratory to effectively address specific issues, that is crucial
for a safety laboratory engineer when designing safety experiments appropriately to analyze thermal risks
Industry perspective on the selection of regulatory starting materials for synthetic peptides
No full textThis perspective from the Synthetic Peptide Working Group of the International Quality Consortium Drug Substance Leadership Group discusses the selection of regulatory starting materials (RSMs) for peptide manufacture. Given the ubiquity of solid phase peptide synthesis (SPPS), it has been common practice to simply default to individual amino acids or dipeptides as RSMs. However, as the field of synthetic peptide research has grown and new synthesis technologies have been more widely adopted, this team proposes that there are cases where significant scientific and technical justification exists to consider larger peptide fragments as RSMs which remain consistent with ICH Q11. This framework would provide greater flexibility and support for the adoption of new and superior peptide synthesis technologies, increasing manufacturing process and supply chain robustness and offering opportunities for industry to address sustainability challenges inherent to current practices in synthetic peptide manufacture
New Inhibitors of β-1,4-Galactosyltransferase I Discovered by Virtual Screening.
No full textSeven different enzymes comprise the galactosyltransferases family, of which β-1,4-galactosyltransferase I (β-1,4-GALT1) is the major contributor to galactosylation activity in cells. Since abnormalities in galactosylation are associated with many pathophysiological conditions, β-1,4-GALT1 is an interesting new target for drug discovery and molecular probe design. There are several known β-1,4-GALT1 inhibitors, but most of them suffer from low cell permeability and thus low in vivo activity. In the present work, we describe an in silico screening performed using commercially available virtual compound libraries that led us to the discovery of novel β-1,4-GALT1 inhibitors. A virtual screening campaign was performed by docking compound libraries to the binding site of β-1,4-GALT1, followed by biological evaluation of selected hits for their β-1,4-GALT1 inhibitory activity. The IC50 values were determined for the best performing inhibitors to obtain new chemotypes of β-1,4-GALT1 inhibitors
Risk factors for barbering in laboratory mice.
No full textBarbering is a common abnormal behavior in laboratory mice, where mice pluck their own fur and/or the fur or whiskers of their cage mates. Barbering mice are a concern for welfare and research quality, as well as serving as a spontaneous model of trichotillomania (a hair-pulling disorder in humans). Causes and prevention of barbering are poorly understood, although there is evidence that both biological and environmental factors play a role in its prevalence. Since initial work in this area was done 20 years ago, mouse husbandry has changed dramatically. We provide an updated analysis of risk factors for barbering in laboratory mice based on point prevalence of hair loss in 2544 cages over one year (7007 mice). We analyzed the effects of biological, environmental, and husbandry factors that are known to be stressors for mice. We found that certain risk factors for barbering, such as sex and breeding status, have persisted despite changes in housing. We additionally identified differences in prevalence based on genetic background, housing system, time of year, and a "hotspot" effect showing spatial clustering of barbering. Our findings can be used to increase understanding of this behavior and to inform changes in husbandry to reduce its prevalence
Induction of proximal tubular proliferation and lengthening in response to sodium glucose linked cotransporter‐2 inhibition in experimental rats
No full textAims/Introduction
While SGLT2 accounts for >90% of kidney glucose reabsorption, its pharmacological inhibition or genetic knockdown reduces glucose reabsorption by only 50%.
Materials and Methods
We postulated that the less than expected glucosuric response to SGLT2 inhibition might result from a compensatory increase in the length of the proximal tubule as seen in experimental diabetes where early tubular proliferation is followed by tubular lengthening. Taking advantage of their differing anatomical locations, stereological techniques were used to differentiate the SGLT1 expressing straight proximal tubule that lies within the outer stripe of the outer medulla (S3 segment) and that of the predominantly SGLT2 expressing early proximal convoluted tubule located within the kidney cortex (S1, S2 segments).
Results
The SGLT2 inhibitor, dapagliflozin, induced an early, transient hyperplastic response (3-fold increase of Ki67 labelling, P < 0.0001) in S3 proximal tubular cells followed by a 32% increase in its length (P < 0.0001). In contrast, the length of the SGLT2 expressing S1, S2 segments of the proximal tubule was unaffected.
Conclusions
The finding that SGLT2 inhibition leads to expansion of the S3 segment of the proximal tubule, the site of SGLT1, is suggestive of a physiological response to diminish urinary glucose loss akin to that occurring in experimental diabetes. These findings provide a cogent explanation for the less-thanthan-expected effect of this drug class on glucose reabsorption
Solvent Sustainability in Drug Discovery: Where Are We Now, and How Can We Improve?
No full textSolvent selection plays a critical role in promoting synthetic chemistry reactions. Metrics from member companies of the ACS Green Chemistry Institute Pharmaceutical Roundtable (ACSGCIPR) have led to an estimate that solvents utilized contribute to 80-90% in terms of the mass of waste streams and 75-80% of the overall life cycle impact of pharmaceutical manufacturing processes. While the bulk of the solvents are typically utilized in development and manufacturing, it has been estimated that up to 3.5 million kilograms of waste are generated from chemistry associated with drug discovery through preclinical studies with the sheer number of chemical reactions performed off-setting the relatively small scale that each reaction is run on. Herein, the medicinal chemistry team of the ACS-GCIPR have performed a systematic study of the most common transformations (over 400k reactions) utilized in the drug discovery phase examining the solvents that these reactions are performed in. Specifically, we have focused on the use of several solvents, which are undesirable based on their hazardous nature and/or known adverse effects on human health. The data has shown that in over 40% of the cases, the reactions profiled are conducted in what is considered to be an undesirable solvent with the use of methylene chloride (DCM) and N,N-dimethylformamide (DMF) being particularly prevalent (notably the most utilized transformation, amide bond formation, is carried out ~ 70% of the time in these solvents). In addition, the methods/solvents used in the workup/purification phases of these reactions was also studied with again a ca. 40% incidence of non-sustainable solvent use being recorded (most notably DCM in both extractions and chromatography). While numerous resources such as solvent guides/selection tools have been developed, and technologies such as High-Throughput Experimentation (HTE) enable solvent screening on sub-mg scales, the continued use of undesirable solvents based on legacy habits in drug discovery campaigns represents a cause for concern from a sustainability perspective. This can also place a significant onus specifically in terms of time pressure on development organizations in having to re-develop chemistry in alternative reaction media when a specific compound progresses towards the clinic
SACF and GILA assays on AML12 cells show limited predictive value for mouse liver genotoxicity.
No full textHepatocellular carcinoma (HCC) has been observed in neonatal mice following the integration of recombinant Adeno-Associated Viruses (rAAV) into the Rian locus. rAAV-related oncogenic risk for patients remains unclear, and the lack of relevant in vitro methods hinders its proper assessment. The soft agar colony-forming (SACF) assay and the growth in low attachment assay (GILA) monitor anchorage-independent growth, a hallmark of transformed adherent cells, and have been previously proposed to assess the tumorigenicity of CRISPR/Cas9-edited human MCF10A cells. Here, we introduce murine versions of SACF and GILA as surrogate in vitro systems to evaluate the risk of HCC development following genome editing or rAAV induced insertional mutagenesis. Selected tumor suppressors linked to HCC onset in vivo were edited through CRISPR/Cas9 in the hepatic murine cell line AML12. The knockout of neurofibromin (Nf2) and the dual inactivation of tumor protein p53 (Tp53) and phosphatase and tensin homolog (Pten) induced anchorage-independence, while the editing of Axin1, Ctnnb1 (coding for β-catenin), and tuberous sclerosis complex 1 (Tsc1) did not promote growth in anchorage-free conditions. Additionally, we generated stable AML12 and MCF10A clones with the rAAV genome respectively integrated into Rian and MEG8, the human homolog of Rian; however, these clones did not show anchorage independence when seeded in SACF and GILA. Overall, the murine SACF and GILA exhibit low predictive value for HCC development, failing to detect rAAV- and tumor-suppressors-associated oncogenicity. While further optimization may improve assays performance, these results highlight the need for more appropriate in vitro methodologies to accurately evaluate rAAV genotoxicity
