Romidepsin (Istodax®), a selective inhibitor of histone deacetylases (HDACs), was approved for the treatment of cutaneous T-cell lymphoma in November 2009 by the U.S. FDA. This unique natural product was discovered from cultures of Chromobacterium violaceum, a Gram-negative bacterium isolated from a Japanese soil sample. This bicyclic compound acts as a prodrug, its disulfide bridge being reduced by glutathione upon uptake into the cell, allowing the free thiol groups to interact with Zn ions in the active site of class I and II histone deacetylase enzymes. Due to the synthetic complexity of the compound, as well as the low yield from the producing organism, analogs are sought to create synthetically accessible alternatives. As a T-cell lymphoma drug, romidepsin offers a valuable new treatment for diseases with few effective therapies.
Variation in the growing environment can have significant impacts on the quantity and diversity of fungal secondary metabolites. In the industrial setting, optimization of growing conditions can lead to significantly increased production of a compound of interest. Such optimization becomes challenging in a drug-discovery screening situation, as the ideal conditions for one organism may induce poor metabolic diversity for a different organism. Here, the impact of different media types, including six liquid media and five solid media, on the secondary metabolite production of three fungal strains was examined in the context of the drug-discovery screening process. The relative production of marker compounds was used to evaluate the usefulness and reliability of each medium for the purpose of producing secondary metabolites.
The use of epigenetic modifiers, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors, has been explored increasingly as a technique to induce the production of additional microbial secondary metabolites. The application of such molecules to microbial cultures has been shown to upregulate otherwise suppressed genes, and in several cases has led to the production of new molecular structures. In this study, the proteasome inhibitor bortezomib was used to induce the production of an additional metabolite from a filamentous fungus (Pleosporales). The induced metabolite was previously isolated from a plant, but the configuration was not assigned until now; in addition, an analogue was isolated from a degraded sample, yielding a new compound. Proteasome inhibitors have not previously been used in this application and offer an additional tool for microbial genome mining.
Introduction
Grapefruit juice can increase or decrease the systemic exposure of myriad oral medications, leading to untoward effects or reduced efficacy. Furanocoumarins in grapefruit juice have been established as inhibitors of cytochrome P450 3A (CYP3A)-mediated metabolism and P-glycoprotein (P-gp)-mediated efflux, while flavonoids have been implicated as inhibitors of organic anion transporting polypeptide (OATP)-mediated absorptive uptake in the intestine. The potential for drug interactions with a food product necessitates an understanding of the expected concentrations of a suite of structurally diverse and potentially bioactive compounds.
Objective
Develop methods for the rapid quantitation of two furanocoumarins (bergamottin and 6′,7′-dihydroxybergamottin) and four flavonoids (naringin, naringenin, narirutin, and hesperidin) in five grapefruit juice products using ultra performance liquid chromatography (UPLC).
Methodology
Grapefruit juice products were extracted with ethyl acetate; the concentrated extract was analyzed by UPLC using acetonitrile:water gradients and a C18 column. Analytes were detected using a photodiode array detector, set at 250 nm (furanocoumarins) and 310 nm (flavonoids). Intraday and interday precision and accuracy and limits of detection and quantitation were determined.
Results
Rapid (<5.0 min) UPLC methods were developed to measure the aforementioned furanocoumarins and flavonoids. R2 values for the calibration curves of all analytes were >0.999. Considerable between-juice variation in the concentrations of these compounds was observed, and the quantities measured were in agreement with the concentrations published in HPLC studies.
Conclusion
These analytical methods provide an expedient means to quantitate key furanocoumarins and flavonoids in grapefruit juice and other foods used in dietary substance-drug interaction studies.
The grapefruit juice-fexofenadine interaction involves inhibition of intestinal organic anion transporting polypeptide (OATP)-mediated uptake. Only naringin has been shown clinically to inhibit intestinal OATP; other constituents have not been evaluated. The effects of a modified grapefruit juice devoid of furanocoumarins (~99%) and polymethoxyflavones (~90%) on fexofenadine disposition were compared to effects of the original juice. Extracts of both juices inhibited estrone 3-sulfate and fexofenadine uptake by similar extents in OATP-transfected cells (~50% and ~25%, respectively). Healthy volunteers (n=18) were administered fexofenadine (120 mg) with water, grapefruit juice, or modified grapefruit juice (240 ml) by randomized, three-way crossover design. Compared to water, both juices decreased fexofenadine geometric mean AUC and Cmax by ~25% (p≤0.008 and p≤0.011, respectively), with no effect on terminal half-life (p=0.11). Similar effects by both juices on fexofenadine pharmacokinetics indicate furanocoumarins and polymethoxyflavones are not major mediators of the grapefruit juice-fexofenadine interaction.
Despite growing popularity in dietary supplements, many medicinal mushrooms have not been evaluated for their safe human consumption using modern techniques. The multifaceted approach described here relies on five key principles to evaluate the safety of non-culinary fungi for human use: (1) identification by sequencing the nuclear ribosomal internal transcribed spacer (ITS) region (commonly referred to as ITS barcoding), (2) screening an extract of each fungal raw material against a database of known fungal metabolites, (3) comparison of these extracts to those prepared from grocery store-bought culinary mushrooms using UHPLCPDA-ELS-HRMS, (4) review of the toxicological and chemical literature for each fungus, and (5) evaluation of data establishing presence in-market. This weight-of-evidence approach was used to evaluate seven fungal raw materials and determine safe human use for each. Such an approach may provide an effective alternative to conventional toxicological animal studies (or more efficiently identifies when studies are necessary) for the safety assessment of fungal dietary ingredients.
Dietary supplements are a multi-billion dollar business, with yearly profit increases. Allegedly safe, these supplements are marketed to a variety of niches, encompassing claims from immune support to weight loss. Six sports nutrition supplements were acquired that were labeled to contain the furanocoumarin(s) bergamottin and/or 6′,7′-dihydroxybergamottin (DHB), both of which are potent irreversible inhibitors of the prominent drug metabolizing enzyme cytochrome P450 3A (CYP3A). Both furanocoumarins are typically present in grapefruit juice, which has been shown to inhibit intestinal CYP3A, perpetrating an increase in the systemic exposure of certain concomitant ‘victim’ drugs. The acquired supplements were analyzed using ultra-performance liquid chromatography coupled to both a photodiode array (PDA) detector and a triple quadrupole mass spectrometer (MS). Contrary to the product labeling, four of the supplements contained no detectable quantities of either furanocoumarin (LOD 0.060 μg/capsule), while two of the supplements contained minimal amounts (one contained 12.13 (± 0.23) μg bergamottin and 65.51 (± 0.64) μg DHB per capsule; the other contained 2.705 (± 0.069) μg bergamottin per capsule and no detectable quantities of DHB). A CYP3A inhibition bioassay was used to assess whether the actual content of the furanocoumarins correlated with CYP3A inhibitory activity. Despite the low amounts of bergamottin and DHB, CYP3A inhibition by the supplements was greater than could be accounted for by the two furanocoumarins. The additional activity suggests the presence of other potent or highly abundant CYP3A inhibitors.
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