Automated sample preparation of protein solid dosage forms: Novel application for the tablet processing workstation.

Biological macromolecule solid dosage forms represent the frontier of orally administered pharmaceuticals. Analysis of these drug products poses new challenges compared to traditional small molecule tablets. In this study we demonstrate the first, to our knowledge, automated Tablet Processing Workstation (TPW) sample preparation of large molecule tablets. Tablets containing a modified version of human insulin were tested for content uniformity and the automated method was successfully validated for recovery, carryover and displayed manual method equivalency in repeatability and in-process stability. Per TPW's ability to process one sample sequentially, the total analysis cycle time is, in fact, increased. In lieu, a net gain in scientist productivity is realized by enabling continuous operation reducing analytical scientist labor time by 71% compared to manually conducted sample preparation.

Characterizing interspecies differences in gastric fluid properties to improve understanding of in vivo oral drug formulation performance.

An in-depth understanding of the properties of gastric fluid(s) prior to an in vivo pharmacokinetic investigation can vastly improve predictions of in vivo performance. Previously, properties of animal and human gastric fluids have been characterized with varying methods. Unfortunately, characterization has often not been thorough, and some properties, such as density and viscosity, have not been reported. Here, human, porcine and canine gastric fluids were harvested and characterized for pH, viscosity, surface tension, density, and osmolarity. We found that the variability of pH and surface tension between dogs was significantly higher than the variability between pigs, and, furthermore, gastric fluids collected from the same canine species (beagles) housed in two different countries (Denmark and China) had surprisingly different pH values. Next, an in vitro dissolution study in diluted gastric fluids from each species was performed using minitablets containing ibuprofen. Human gastric fluids and porcine gastric fluids showed similar dissolution profiles and corroborated well with biorelevant human Fasted State Simulated Gastric Fluid (FaSSGF). In contrast, differences in canine gastric fluids caused highly variable dissolution results. We systematically compared our findings to those in the literature and based on this evaluation, propose obtaining aspirates from the animals used for in vivo studies to ensure knowledge on the fluid properties affecting the performance of the formulated drug in question.

Macrocyclic Prodrugs of a Selective Nonpeptidic Direct Thrombin Inhibitor Display High Permeability, Efficient Bioconversion but Low Bioavailability.

The only oral direct thrombin inhibitors that have reached the market, ximelagatran and dabigatran etexilat, are double prodrugs with low bioavailability in humans. We have evaluated an alternative strategy: the preparation of a nonpeptidic, polar direct thrombin inhibitor as a single, macrocyclic esterase-cleavable (acyloxy)alkoxy prodrug. Two homologous prodrugs were synthesized and displayed high solubilities and Caco-2 cell permeabilities, suggesting high absorption from the intestine. In addition, they were rapidly and completely converted to the active zwitterionic thrombin inhibitor in human hepatocytes. Unexpectedly, the most promising prodrug displayed only moderately higher oral bioavailability in rat than the polar direct thrombin inhibitor, most likely due to rapid metabolism in the intestine or the intestinal wall. To the best of our knowledge, this is the first in vivo ADME study of macrocyclic (acyloxy)alkoxy prodrugs, and it remains to be established if the modest increase in bioavailability is a general feature of this category of prodrugs or not.

Bivalent histone modifications in stem cells poise miRNA loci for CpG island hypermethylation in human cancer.

It has been proposed that the existence of stem cell epigenetic patterns confer a greater likelihood of CpG island hypermethylation on tumor suppressor-coding genes in cancer. The suggested mechanism is based on the Polycomb-mediated methylation of K27 of histone H3 and the recruitment of DNA methyltransferases on the promoters of tumor suppressor genes in cancer cells, when those genes are preferentially pre-marked in embryonic stem cells (ESCs) with bivalent chromatin domains. On the other hand, miRNAs appear to be dysregulated in cancer, with many studies reporting silencing of miRNA genes due to aberrant hypermethylation of their promoter regions. We wondered whether a pre-existing histone modification profile in stem cells might also contribute to the DNA methylation-associated silencing of miRNA genes in cancer. To address this, we examined a group of tumor suppressor miRNA genes previously reported to become hypermethylated and inactivated specifically in cancer cells. We analyzed the epigenetic events that take place along their promoters in human embryonic stem cells and in transformed cells. Our results suggest that there is a positive correlation between the existence of bivalent chromatin domains on miRNA promoters in ESCs and the hypermethylation of those genes in cancer, leading us to conclude that this epigenetic mark could be a mechanism that prepares miRNA promoters for further DNA hypermethylation in human tumors.