16 research outputs found
Currently favored sampling practices for tumor sequencing can produce optimal results in the clinical setting
Tumor heterogeneity is a consequence of clonal evolution, resulting in a fractal-like architecture with spatially separated main clones, sub-clones and single-cells. As sequencing an entire tumor is not feasible, we ask the question whether there is an optimal clinical sampling strategy that can handle heterogeneity and hypermutations? Here, we tested the effect of sample size, pooling strategy as well as sequencing depth using whole-exome sequencing of ovarian tumor specimens paired with normal blood samples. Our study has an emphasis on clinical application—hence we compared single biopsy, combined local biopsies and combined multi-regional biopsies. Our results show that sequencing from spatially neighboring regions show similar genetic compositions, with few private mutations. Pooling samples from multiple distinct regions of the primary tumor did not increase the overall number of identified mutations but may increase the robustness of detecting clonal mutations. Hypermutating tumors are a special case, since increasing sample size can easily dilute sub-clonal private mutations below detection thresholds. In summary, we compared the effects of sampling strategies (single biopsy, multiple local samples, pooled global sample) on mutation detection by next generation sequencing. In view of the limitations of present tools and technologies, only one sequencing run per sample combined with high coverage (100–300 ×) sequencing is affordable and practical, regardless of the number of samples taken from the same patient. © 2020, The Author(s)
Drug Salt Formation via Mechanochemistry: The Case Study of Vincamine
The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.In the present research a salt of vincamine, a
poorly bioavailable indole alkaloid derived from the leaves of
Vinca minor L., was synthesized in the solid state by means of a
mechanochemical process employing citric acid as a reagent.
The mechanochemical process was adopted as a solvent-free
alternative to classical citrate synthetic route that involves the
use of solvents. Since the mechanochemical salification is little
studied to date and presents the disadvantage of offering a low
yield, in this work, the influence of three process and
formulation variables on the percentage of vincamine citrate
was studied. In particular, the time of mechanical treatment (in
planetary mill Fritsch P5) and the amount of citric acid were
varied in order to evaluate their effect on the yield of the process, and the introduction of a solid solvent, a common
pharmaceutical excipient (sodium carboxymethylcellulose, NaCMC), was considered. Due to the complexity of the resulting
samples’ matrix, an appropriate experimental design was employed to project the experimental trials and the influence of the
three variables on the experimental response was estimated with the help of a statistical analysis. The experimental response, that
is, the yield of the process corresponding to the percentage of vincamine in the protonated form, was unconventionally calculated
by means of X-ray photoelectron spectroscopy analysis (XPS). Out of 16 samples, the one with the highest yield was the
coground sample containing vincamine and citric acid in a 1:2 molar ratio, treated for 60 min in the presence of NaCMC. Under
the above conditions the salification reaction was completed highlighting the importance of a proper selection of process and
formulation variables of the mechanochemical salification, and emphasizing the crucial role of the solid solvent in facilitating the
salification. The second step of the research encompassed the characterization of the citrate salt obtained by solid excipient
assisted mechanochemical salification (SEAMS) in comparison with the vincamine citrate obtained by classical synthetic route.
The samples were characterized by, besides XPS, high resolution transmission electron microscopy (HRTEM), X-ray powder
diffraction (XRPD), in vitro solubilization kinetics and in vivo oral pilot study in rats. Finally, in order to monitor over time
possible disproportionation phenomena, stability studies have been performed by repeating XPS analysis after 8 months. As
expected, the the SEAMS-vincamine salt consisted of particles both crystalline and amorphous. The solubilization kinetics was
superior to the corresponding salt probably thanks to the favorable presence of the hydrophilic excipient although the two salts
were bioequivalent in rats after oral administration. Furthermore, no evidence of disporportionation phenomena in the SEAMSvincamine
salt was found after storage. In conclusion, in the case of forming salts of poorly soluble drugs, the SEAMS process
may be an interesting alternative to both classical synthetic routes, eliminating the need for solvent removal, and simple neat
mechanochemical salification, overcoming the problem of limited process yield