FV-162 is a novel, orally bioavailable, irreversible proteasome inhibitor with improved pharmacokinetics displaying preclinical efficacy with continuous daily dosing.

Approved proteasome inhibitors have advanced the treatment of multiple myeloma but are associated with serious toxicities, poor pharmacokinetics, and most with the inconvenience of intravenous administration. We therefore sought to identify novel orally bioavailable proteasome inhibitors with a continuous daily dosing schedule and improved therapeutic window using a unique drug discovery platform. We employed a fluorine-based medicinal chemistry technology to synthesize 14 novel analogs of epoxyketone-based proteasome inhibitors and screened them for their stability, ability to inhibit the chymotrypsin-like proteasome, and antimyeloma activity in vitro. The tolerability, pharmacokinetics, pharmacodynamic activity, and antimyeloma efficacy of our lead candidate were examined in NOD/SCID mice. We identified a tripeptide epoxyketone, FV-162, as a metabolically stable, potent proteasome inhibitor cytotoxic to human myeloma cell lines and primary myeloma cells. FV-162 had limited toxicity and was well tolerated on a continuous daily dosing schedule. Compared with the benchmark oral irreversible proteasome inhibitor, ONX-0192, FV-162 had a lower peak plasma concentration and longer half-life, resulting in a larger area under the curve (AUC). Oral FV-162 treatment induced rapid, irreversible inhibition of chymotrypsin-like proteasome activity in murine red blood cells and inhibited tumor growth in a myeloma xenograft model. Our data suggest that oral FV-162 with continuous daily dosing schedule displays a favorable safety, efficacy, and pharmacokinetic profile in vivo, identifying it as a promising lead for clinical evaluation in myeloma therapy.

Inhibition of intracellular dipeptidyl peptidases 8 and 9 enhances parthenolide's anti-leukemic activity.

Parthenolide is selectively toxic to leukemia cells; however, it also activates cell protective responses that may limit its clinical application. Therefore, we sought to identify agents that synergistically enhance parthenolide's cytotoxicity. Using a high-throughput combination drug screen, we identified the anti-hyperglycemic, vildagliptin, which synergized with parthenolide to induce death of the leukemia stem cell line, TEX (combination index (CI)=0.36 and 0.16, at effective concentration (EC) 50 and 80, respectively; where CI <1 denotes statistical synergy). The combination of parthenolide and vildagliptin reduced the viability and clonogenic growth of cells from acute myeloid leukemia patients and had limited effects on the viability of normal human peripheral blood stem cells. The basis for synergy was independent of vildagliptin's primary action as an inhibitor of dipeptidyl peptidase (DPP) IV. Rather, using chemical and genetic approaches we demonstrated that the synergy was due to inhibition of the related enzymes DPP8 and DPP9. In summary, these results highlight DPP8 and DPP9 inhibition as a novel chemosensitizing strategy in leukemia cells. Moreover, these results suggest that the combination of vildagliptin and parthenolide could be useful for the treatment of leukemia.

Regulation and expression of multidrug resistance (MDR) transcripts in the intestinal epithelium.

A paucity of information exists on the regulation of gene expression in the undifferentiated intestine. The intestinal epithelium is one of the few normal tissues expressing the multidrug resistance (MDR) genes that confer the multidrug resistant phenotype to a variety of tumours. Expression of mdr1a has been observed in the primitive rat intestinal epithelial cell line, IEC-18. It is hypothesized that characterization of MDR gene expression in IEC-18 cells will provide insight into gene regulation in undifferentiated intestinal cells. A series of hamster mdr1a promoter deletion constructs was studied in IEC-18 and a region with 12-13-fold enhancer activity was identified. This region was shown to function in an orientation- and promoter context-independent manner, specifically in IEC-18 cells. Unexpectedly, Northern probing revealed a greater expression of mdr1b than mdr1a in IEC-18 cells. A quantitative reverse transcription polymerase chain reaction assay was used to compare the relative expression of MDR genes in IEC cells, fetal intestine, and in the undifferentiated and differentiated components of adult intestinal epithelium. MDR transcript levels in IEC cells were found to resemble those of fetal intestine and small intestinal crypts, where a conversion from mixed mdr1a/mdr1b to predominantly mdr1a expression occurs as cells mature. This work describes two contributions to the field of gene regulation in the undifferentiated intestine--first, the initial characterization of a putative mdr1a enhancer region with specificity for primitive intestinal cells and secondly, the first report of mdr1b detection in the intestine and its expression in primitive cell types.

Evidence for light-dependent recycling of respired carbon dioxide by the cotton fruit.

Conservation of respired CO(2) by an efficient recycling mechanism in fruit could provide a significant source of C for yield productivity. However, the extent to which such a mechanism operates in cotton (Gossypium hirsutum L.) is unknown. Therefore, a combination of CO(2) exchange, stable C isotope, and chlorophyll (Chl) fluorescence techniques were used to examine the recycling of respired CO(2) in cotton fruit. Respiratory CO(2) losses of illuminated fruit were reduced 15 to 20% compared with losses for dark-incubated fruit. This light-dependent reduction in CO(2) efflux occurred almost exclusively via the fruit's outer capsule wall. Compared with the photosynthetic activity of leaves, CO(2) recycling by the outer capsule wall was 35 to 40% as efficient. Calculation of (14)CO(2) fixation on a per Chl basis revealed that the rate of CO(2) recycling for the capsule wall was 62.2 micromoles (14)CO(2) per millimole Chl per second compared with an assimilation rate of 64.6 micromoles (14)CO(2) per millimole Chl per second for leaves. During fruit development, CO(2) recycling contributed more than 10% of that C necessary for fruit dry weight growth. Carbon isotope analyses (delta(13)C) showed significant differences among the organs examined, but the observed isotopic compositions were consistent with a C(3) pathway of photosynthesis. Pulse-modulated Chl fluorescence indicated that leaves and fruit were equally efficient in photochemical and nonphotochemical dissipation of light energy. These studies demonstrated that the cotton fruit possesses a highly efficient, light-dependent CO(2) recovery mechanism that aids in the net retention of plant C and, therein, contributes to yield productivity.