Evaluation of a Novel Renewable Hepatic Cell Model for Prediction of Clinical CYP3A4 Induction Using a Correlation-Based Relative Induction Score Approach

ABSTRACT Metabolism enzyme induction-mediated drug-drug interactions need to be carefully characterized in vitro for drug candidates to predict in vivo safety risk and therapeutic efficiency. Currently, both the Food and Drug Administration and European Medicines Agency recommend using primary human hepatocytes as the gold standard in vitro test system for studying the induction potential of candidate drugs on cytochrome P450 (CYP), CYP3A4, CYP1A2, and CYP2B6. However, primary human hepatocytes are known to bear inherent limitations such as limited supply and large lot-to-lot variations, which result in an experimental burden to qualify new lots. To overcome these shortcomings, a renewable source of human hepatocytes (i.e., Corning HepatoCells) was developed from primary human hepatocytes and was evaluated for in vitro CYP3A4 induction using methods well established by the pharmaceutical industry. HepatoCells have shown mature hepatocyte-like morphology and demonstrated primary hepatocyte-like response to prototypical inducers of all three CYP enzymes with excellent consistency. Importantly, HepatoCells retain a phenobarbital-responsive nuclear translocation of human constitutive androstane receptor from the cytoplasm, characteristic to primary hepatocytes. To validate HepatoCells as a useful tool to predict potential clinical relevant CYP3A4 induction, we tested three different lots of HepatoCells with a group of clinical strong, moderate/weak CYP3A4 inducers, and noninducers. A relative induction score calibration curve-based approach was used for prediction. HepatoCells showed accurate prediction comparable to primary human hepatocytes. Together, these results demonstrate that Corning HepatoCells is a reliable in vitro model for drug-drug interaction studies during the early phase of drug testing

Corrosion control using regenerative biofilms and biofilm inhibition using plant-derived chemicals

Bacteria form biofilms on any surface and dominate as sessile cells in most ecosystems. Deleterious biofilms cause problems in both industry (e.g., corrosion of metals) and in medicine (e.g., infections on human organs), which makes it necessary to develop novel strategies to control corrosion and deleterious biofilms. ^ Two strategies are being investigated to control biocorrosion and deleterious biofilms. First, regenerative beneficial biofilms are used to protect metals. Biofilms producing gramicidin S [a 10-residue, cyclic peptide with the structure of cyclo(-Val-Orn-Leu-D-Phe-Pro-)2] were able to protect mild steel 1010 by inhibiting corrosion-causing, sulfate-reducing bacteria (SRB) in the aggressive, cooling service water of the AmerGen Three-Mile-Island (TMI) nuclear plant. A gramicidin-S-producing Bacillus brevis 18-3 biofilm protected mild steel by inhibiting both D. orientis and L. discophora SP-6 simultaneously. ^ Another strategy is to discover natural compounds that can inhibit biofilms without causing resistance to antibiotics. A new biofilm inhibitor, ursolic acid, inhibits biofilms of multiple bacteria without affecting growth and is effective in various media. As predicted by the DNA microarrays studying the gene expression profiles of E. coli K12 ATCC25404 grown with and without ursolic acid, removing motAB counteracts the inhibition of ursolic acid (which induces expression of motAB to make cells too motile). It was also discovered that sulfur metabolism (through cysB) affects biofilm formation in the absence of ursolic acid. ^ Mutant with both hha (a gene encoding for an environmental-response regulator of the pathogenic hemolysin operon) and its contiguous gene ybaJ deleted (Δhha/ΔybaJ) were tested for biofilm formation. It appears Hha and YbaJ increase biofilm formation by decreasing motility when a conjugative plasmid (R1drd 19) is present and that YbaJ plays the most important role in this regulation of motility. When hha/ybaJ is deleted, there is less conjugation, more motility and less biofilm. ^ In addition to ursolic acid, palmitoleic acid and asiatic acid also show biofilm inhibition without affecting growth rates. It is first presented here that a synergic effect is found for palmitoleic acid and the hha/ybaJ mutant in terms of biofilm inhibition. Analogs of ursolic acid showed comparable biofilm inhibition ability with asiatic acid possesses even stronger inhibition ability.

Autoinducer 2 Controls Biofilm Formation in Escherichia coli through a Novel Motility Quorum-Sensing Regulator (MqsR, B3022)

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene)

Differential Gene Expression for Investigation of Escherichia coli Biofilm Inhibition by Plant Extract Ursolic Acid

After 13,000 samples of compounds purified from plants were screened, a new biofilm inhibitor, ursolic acid, has been discovered and identified. Using both 96-well microtiter plates and a continuous flow chamber with COMSTAT analysis, 10 μg of ursolic acid/ml inhibited Escherichia coli biofilm formation 6- to 20-fold when added upon inoculation and when added to a 24-h biofilm; however, ursolic acid was not toxic to E. coli, Pseudomonas aeruginosa, Vibrio harveyi, and hepatocytes. Similarly, 10 μg of ursolic acid/ml inhibited biofilm formation by >87% for P. aeruginosa in both complex and minimal medium and by 57% for V. harveyi in minimal medium. To investigate the mechanism of this nontoxic inhibition on a global genetic basis, DNA microarrays were used to study the gene expression profiles of E. coli K-12 grown with or without ursolic acid. Ursolic acid at 10 and 30 μg/ml induced significantly (P < 0.05) 32 and 61 genes, respectively, and 19 genes were consistently induced. The consistently induced genes have functions for chemotaxis and mobility (cheA, tap, tar, and motAB), heat shock response (hslSTV and mopAB), and unknown functions (such as b1566 and yrfHI). There were 31 and 17 genes repressed by 10 and 30 μg of ursolic acid/ml, respectively, and 12 genes were consistently repressed that have functions in cysteine synthesis (cysK) and sulfur metabolism (cysD), as well as unknown functions (such as hdeAB and yhaDFG). Ursolic acid inhibited biofilms without interfering with quorum sensing, as shown with the V. harveyi AI-1 and AI-2 reporter systems. As predicted by the differential gene expression, deleting motAB counteracts ursolic acid inhibition (the paralyzed cells no longer become too motile). Based on the differential gene expression, it was also discovered that sulfur metabolism (through cysB) affects biofilm formation (in the absence of ursolic acid)