Development of ADS051, an oral, gut-restricted, small molecule neutrophil modulator for the treatment of neutrophil-mediated inflammatory diseases.

Neutrophils are an essential component of the innate immune system; however, uncontrolled neutrophil activity can lead to inflammation and tissue damage in acute and chronic diseases. Despite inclusion of neutrophil presence and activity in clinical evaluations of inflammatory diseases, the neutrophil has been an overlooked therapeutic target. The goal of this program was to design a small molecule regulator of neutrophil trafficking and activity that fulfilled the following criteria: (a) modulates neutrophil epithelial transmigration and activation, (b) lacks systemic exposure, (c) preserves protective host immunity, and (d) is administered orally. The result of this discovery program was ADS051 (also known as BT051), a low permeability, small molecule modulator of neutrophil trafficking and activity via blockade of multidrug resistance protein 2 (MRP2)- and formyl peptide receptor 1 (FPR1)-mediated mechanisms. ADS051, based on a modified scaffold derived from cyclosporine A (CsA), was designed to have reduced affinity for calcineurin with low cell permeability and, thus, a greatly reduced ability to inhibit T-cell function. In cell-based assays, ADS051 did not inhibit cytokine secretion from activated human T cells. Furthermore, in preclinical models, ADS051 showed limited systemic absorption (<1% of total dose) after oral administration, and assessment of ADS051 in human, cell-based systems demonstrated inhibition of neutrophil epithelial transmigration. In addition, preclinical toxicology studies in rats and monkeys receiving daily oral doses of ADS051 for 28 days did not reveal safety risks or ADS051-related toxicity. Our results to date support the clinical development of ADS051 in patients with neutrophil-mediated inflammatory diseases.

Systemic ASBT inactivation protects against liver damage in obstructive cholestasis in mice.

Background & Aims: Non-absorbable inhibitors of the apical sodium-dependent bile acid transporter (ASBT; also called ileal bile acid transporter [IBAT]) are recently approved or in clinical development for multiple cholestatic liver disorders and lead to a reduction in pruritus and (markers for) liver injury. Unfortunately, non-absorbable ASBT inhibitors (ASBTi) can induce diarrhoea or may be ineffective if cholestasis is extensive and largely precludes intestinal excretion of bile acids. Systemically acting ASBTi that divert bile salts towards renal excretion may alleviate these issues. Methods: Bile duct ligation (BDL) was performed in ASBT-deficient (ASBT knockout [KO]) mice as a model for chronic systemic ASBT inhibition in obstructive cholestasis. Co-infusion of radiolabelled taurocholate and inulin was used to quantify renal bile salt excretion after BDL. In a second (wild-type) mouse model, a combination of obeticholic acid (OCA) and intestine-restricted ASBT inhibition was used to lower the bile salt pool size before BDL. Results: After BDL, ASBT KO mice had reduced plasma bilirubin and alkaline phosphatase compared with wild-type mice with BDL and showed a marked reduction in liver necrotic areas at histopathological analysis, suggesting decreased BDL-induced liver damage. Furthermore, ASBT KO mice had reduced bile salt pool size, lower plasma taurine-conjugated polyhydroxylated bile salt, and increased urinary bile salt excretion. Pretreatment with OCA + ASBTi in wild-type mice reduced the pool size and greatly improved liver injury markers and liver histology. Conclusions: A reduced bile salt pool at the onset of cholestasis effectively lowers cholestatic liver injury in mice. Systemic ASBT inhibition may be valuable as treatment for cholestatic liver disease by lowering the pool size and increasing renal bile salt output even under conditions of minimal faecal bile salt secretion. Lay summary: Novel treatment approaches against cholestatic liver disease (resulting in reduced or blocked flow of bile) involve non-absorbable inhibitors of the bile acid transport protein ASBT, but these are not always effective and/or can cause unwanted side effects. In this study, we demonstrate that systemic inhibition/inactivation of ASBT protects mice against developing severe cholestatic liver injury after bile duct ligation, by reducing bile salt pool size and increasing renal bile salt excretion.

Pacific Blue-Taxoids as Fluorescent Molecular Probes of Microtubules.

Taxoids such as paclitaxel (Taxol) are an important class of anticancer drugs that bind ß-tubulin and stabilize cellular microtubules. To provide new chemical tools for studies of microtubules, we synthesized derivatives of paclitaxel modified at the 7-position with the small coumarin-derived fluorophore Pacific Blue (PB). Three of these Pacific Blue-Taxoids termed PB-Gly-Taxol, PB-ß-Ala-Taxol, and PB-GABA-Taxol bind purified crosslinked microtubules with affinities of 34-265 nM, where the affinity can be tuned based on the length of an amino acid linker. When added to living cells in the presence of verapamil or probenecid as inhibitors of efflux, these compounds allow visualization of the microtubule network by confocal microscopy. We describe methods for the synthesis of these probes, determination of their affinities for crosslinked tubulin, and imaging of microtubules in living HeLa cells. We further describe their uptake by Caco-2 cells and two transporter-deficient Caco-2 knockout cell lines in the absence and presence of efflux inhibitors by flow cytometry. These studies revealed that p-glycoprotein (MDR1) and multidrug-resistance protein 2 (MRP2) are major mediators of efflux of these molecular probes. These compounds provide useful tools for studies of microtubules and cellular efflux transporters in living cells.

Biliary excretion of arsenic by human HepaRG cells is stimulated by selenide and mediated by the multidrug resistance protein 2 (MRP2/ABCC2).

Millions of people worldwide are exposed to unacceptable levels of arsenic, a proven human carcinogen, in drinking water. In animal models, arsenic and selenium are mutually protective through formation and biliary excretion of seleno-bis (S-glutathionyl) arsinium ion [(GS)2AsSe]-. Selenium-deficient humans living in arsenic-endemic regions are at increased risk of arsenic-induced diseases, and may benefit from selenium supplementation. The influence of selenium on human arsenic hepatobiliary transport has not been studied using optimal human models. HepaRG cells, a surrogate for primary human hepatocytes, were used to investigate selenium (selenite, selenide, selenomethionine, and methylselenocysteine) effects on arsenic hepatobiliary transport. Arsenite + selenite and arsenite + selenide at different molar ratios revealed mutual toxicity antagonism, with the latter being higher. Significant levels of arsenic biliary excretion were detected with a biliary excretion index (BEI) of 14 ± 8%, which was stimulated to 32 ± 7% by selenide. Consistent with the formation and biliary efflux of [(GS)2AsSe]-, arsenite increased the BEI of selenide from 0% to 24 ± 5%. Arsenic biliary excretion was lost in the presence of selenite, selenomethionine, and methylselenocysteine. Sinusoidal export of arsenic was stimulated ∼1.6-fold by methylselenocysteine, but unchanged by other selenium forms. Arsenic canalicular and sinusoidal transport (±selenide) was temperature- and GSH-dependent and inhibited by MK571. Knockdown experiments revealed that multidrug resistance protein 2 (MRP2/ABCC2) accounted for all detectable biliary efflux of arsenic (±selenide). Overall, the chemical form of selenium and human MRP2 strongly influenced arsenic hepatobiliary transport, information critical for human selenium supplementation in arsenic-endemic regions.

3D organoids derived from the small intestine: An emerging tool for drug transport research.

Small intestine in vitro models play a crucial role in drug transport research. Although conventional 2D cell culture models, such as Caco-2 monolayer, possess many advantages, they should be interpreted with caution because they have relatively poor physiologically reproducible phenotypes and functions. With the development of 3D culture technology, pluripotent stem cells (PSCs) and adult somatic stem cells (ASCs) show remarkable self-organization characteristics, which leads to the development of intestinal organoids. Based on previous studies, this paper reviews the application of intestinal 3D organoids in drug transport mediated by P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2). The advantages and limitations of this model are also discussed. Although there are still many challenges, intestinal 3D organoid model has the potential to be an excellent tool for drug transport research.

Expression and phylogeny of multidrug resistance protein 2 and 4 in African white backed vulture (Gyps africanus).

Diclofenac toxicity in old world vultures is well described in the literature by both the severity of the toxicity induced and the speed of death. While the mechanism of toxicity remains unknown at present, the necropsy signs of gout suggests primary renal involvement at the level of the uric acid excretory pathways. From information in the chicken and man, uric acid excretion is known to be a complex process that involves a combination of glomerular filtration and active tubular excretion. For the proximal convoluted tubules excretion occurs as a two-step process with the basolateral cell membrane using the organic anion transporters and the apical membrane using the multidrug resistant protein to transport uric acid from the blood into the tubular fluid. With uric acid excretion seemingly inhibited by diclofenac, it becomes important to characterize these transporter mechanism at the species level. With no information being available on the molecular characterization/expression of MRPs of Gyps africanus, for this study we used next generation sequencing, and Sanger sequencing on the renal tissue of African white backed vulture (AWB), as the first step to establish if the MRPs gene are expressed in AWB. In silico analysis was conducted using different software to ascertain the function of the latter genes. The sequencing results revealed that the MRP2 and MRP4 are expressed in AWB vultures. Phylogeny of avian MRPs genes confirms that vultures and eagles are closely related, which could be attributed to having the same ancestral genes and foraging behavior. In silico analysis confirmed the transcribed proteins would transports anionic compounds and glucose.

Breast Cancer Resistance Protein and Multidrug Resistance Protein 2 Mediate the Disposition of Leonurine-10-O-ß-glucuronide.

BACKGROUND: Leonurine (Leo), a promising antilipemic agent that has been approved for clinical trials, is extensively metabolized into bioactive Leonurine-10-O-ß-glucuronide (L-10-G) vivo. OBJECTIVE: To explore the effects of breast cancer resistance protein (Bcrp) and multidrug resistance protein 2 (Mrp2) on the disposition of L-10-G. METHODS: The pharmacokinetics, tissue distribution and intestinal perfusion of Leo were studied by using efflux transporter gene knockout mouse models. The enzyme kinetics via liver and intestinal microsomes were also examined. RESULTS: After intravenous injection with Leo, the AUC0-∞ values of L-10-G in Bcrp1-/- and Mrp2-/- mice were 1.55-fold and 16.80-fold higher, respectively, than those in wild-type FVB mice (P < 0.05). After oral administration, the AUC0-∞ value of L-10-G showed a 2.82-fold increase in Mrp2-/- mice compared with wild-type FVB mice (P < 0.05). After gavage with Leo for 10 and 25 min, the bile accumulation of L-10-G in Mrp2-/- mice was 3-fold and 22-fold lower, respectively, than that in wild-type FVB mice (P < 0.05). Besides, the intestinal excreted amount of L-10-G showed 2.22-fold and 2.68-fold decrease in Bcrp1-/- and Mrp2-/- mice, respectively, compared with that in wild-type FVB mice (P < 0.05). The clearance of L-10-G decreased in liver microsomes and increased in intestinal microsomes of Bcrp1-/- and Mrp2-/- mice compared to the wild-type FVB mice (P < 0.05). CONCLUSION: Both Bcrp and Mrp2 are involved in the disposition of L-10-G, and Mrp2 exhibits a superior influence.

Endoplasmic Reticulum Stress Increases Multidrug-resistance Protein 2 Expression and Mitigates Acute Liver Injury.

BACKGROUND: Multidrug-resistance protein (MRP) 2 is a key membrane transporter that is expressed on hepatocytes and regulated by nuclear factor kappa B (NF-κB). Interestingly, endoplasmic reticulum (ER) stress is closely associated with liver injury and the activation of NF-κB signaling. OBJECTIVE: Here, we investigated the impact of ER stress on MRP2 expression and the functional involvement of MRP2 in acute liver injury. METHODS: ER stress, MRP2 expression, and hepatocyte injury were analyzed in a carbon tetrachloride (CCl4)-induced mouse model of acute liver injury and in a thapsigargin (TG)-induced model of ER stress. RESULTS: CCl4 and TG induced significant ER stress, MRP2 protein expression and NF- κB activation in mice and LO2 cells (P < 0.05). Pretreatment with ER stress inhibitor 4- phenyl butyric acid (PBA) significantly mitigated CCl4 and TG-induced ER stress and MRP2 protein expression (P < 0.05). Moreover, pretreatment with pyrrolidine dithiocarbamic acid (PDTC; NF-κB inhibitor) significantly inhibited CCl4-induced NF-κB activation and reduced MRP2 protein expression (1±0.097 vs. 0.623±0.054; P < 0.05). Furthermore, hepatic downregulation of MRP2 expression significantly increased CCl4- induced ER stress, apoptosis, and liver injury. CONCLUSION: ER stress enhances intrahepatic MRP2 protein expression by activating NF-κB. This increase in MRP2 expression mitigates ER stress and acute liver injury.

Transport-Mediated Oxaliplatin Resistance Associated with Endogenous Overexpression of MRP2 in Caco-2 and PANC-1 Cells.

Our recent publications showed that multidrug resistance protein 2 (MRP2, encoded by the ABCC2 gene) conferred oxaliplatin resistance in human liver cancer HepG2 cells. However, the contribution of MRP2 to oxaliplatin resistance remains unclear in colorectal and pancreatic cancer lines. We investigated the effects of silencing MRP2 by siRNA on oxaliplatin accumulation and sensitivity in human colorectal cancer Caco-2 cells and pancreatic cancer PANC-1 cells. We characterized the effects of oxaliplatin on MRP2 ATPase activities using membrane vesicles. Over-expression of MRP2 (endogenously in Caco-2 and PANC-1 cells) was associated with decreased oxaliplatin accumulation and cytotoxicity, but those deficits were reversed by inhibition of MRP2 with myricetin or siRNA knockdown. Silencing MRP2 by siRNA increased oxaliplatin-induced apoptotic rate in Caco-2 and PANC-1 cells. Oxaliplatin stimulated MRP2 ATPase activity with a concentration needed to reach 50% of the maximal stimulation (EC50) value of 8.3 ± 0.7 µM and Hill slope 2.7. In conclusion, oxaliplatin is a substrate of MRP2 with possibly two binding sites, and silencing MRP2 increased oxaliplatin accumulation and cytotoxicity in two widely available gastrointestinal tumour lines (PANC-1 and Caco-2).

Dysregulation of BSEP and MRP2 May Play an Important Role in Isoniazid-Induced Liver Injury via the SIRT1/FXR Pathway in Rats and HepG2 Cells.

To explore the role of the abnormal expression of the bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in isoniazid (INH)-induced liver injury, we assessed the liver injury induced by INH in rats and HepG2 cells in vitro. The regulatory pathways via Sirtuin 1 (SIRT1) and farnesoid X receptor (FXR) were also determined. Rat liver injury was assessed by histopathological and biochemical analysis and HepG2 cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. The levels of protein were determined by Western blot. The results indicated that INH could induce hepatotoxicity in vivo and in vitro in a dose dependent manner. The liver index and serum biochemical analysis, especially the levels of total bile acids (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL), were significantly increased in rats. The INH hepatotoxicity was severe in the high dose group, and occurred alongside the down-regulation of BSEP and MRP2 in vivo and in vitro, leading to the accumulation of toxic substrates in the hepatocytes. The SIRT1/FXR pathway was identified as being important for the down-regulation of transporters. In summary, our study indicated that the down-regulation of BSEP and MRP2 represents one mechanism of INH-induced liver injury and the down-regulation of SIRT1/FXR may be a key regulator. This will inform the development of novel therapies and enable the prevention of INH-induced liver injury.

SMS regulates the expression and function of P-gp and MRP2 in Caco-2 cells.

Sphingomyelin synthase (SMS) has two isoforms of SMS1 and SMS2, the last enzyme involved in the biosynthesis of sphingomyelin (SM), and has impact on the expression of membrane proteins. In the present study, we explored the potential effects of SMS on drug transporters, a special family of membrane proteins in human intestinal epithelial Caco-2 cells. The specific knockdown of SMS1 or SMS2 with siRNA in Caco-2 cells substantially decreased the expression and function of P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2) rather than other drug transporters MRP1, MRP3, PEPT1, OATP2B1, and BCRP. In the SMS1 stable overexpressed Caco-2 cell line, the expression levels of P-gp and MRP2 and transcription factor pregnane X receptor (PXR) were upregulated and the phosphorylation levels of signaling pathways janus protein tyrosine kinase 2 (JAK-2) and extracellular signal-regulated kinases (ERK) were also evidently increased; however, the upregulated mRNA expression levels of PXR, P-gp, and MRP2 were diminished by inhibiting the phosphorylation of ERK and JAK-2. Furthermore, the SMS1 overexpression in Caco-2 cells altered the expression levels of ERM proteins ezrin and moesin, which are closely connected to the function of drug transporters. In conclusion, we herein demonstrate for the first time that in Caco-2 cells SMS regulates the expression and function of drug transporters P-gp and MRP2, and their regulator PXR is mediated by phosphorylated ERK and JAK-2 signaling pathways.

Geniposidic acid protected against ANIT-induced hepatotoxity and acute intrahepatic cholestasis, due to Fxr-mediated regulation of Bsep and Mrp2.

ETHNOPHARMACOLOGICAL RELEVANCE: Geniposidic acid (GPA) is the main constituent of Gardenia jasminoides Ellis (Rubiaceae), which has long been used to treat inflammation, jaundice and hepatic disorders. The cholagogic effect of Gardenia jasminoides Ellis (Rubiaceae) and GPA have been widely reported, but the underlying occurrence mechanism remains unclear. AIM OF THE STUDY: This investigation was designed to evaluate the hepatoprotection effect and potential mechanisms of GPA derived from Gardenia jasminoides Ellis (Rubiaceae) on fighting against α-naphthylisothiocyanate (ANIT) caused liver injury with acute intrahepatic cholestasis. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were intragastrically (i.g.) administered with the GPA (100, 50 and 25mg/kg B.W. every 24h) for seven consecutive days, and then they were treated with ANIT (i.g. 65mg/kg once in the 5th day) which induced liver injury with acute intrahepatic cholestasis. Serum and bile biochemical analysis, bile flow rate and liver histopathology were measured to evaluate the protective effect of GPA fight against ANIT treatment. The protein and mRNA expression levels of farnesoid X receptor (Fxr), bile-salt export pump (Bsep), multidrug resistance associated protein2 (Mrp2), were evaluated to study the effect of liver protection about GPA against ANIT induced hepatotoxicity and underlying mechanisms. RESULTS: Some abnormalities were observed on ANIT treated rats including weight loss, reduced food intake and hair turned yellow. Obtained results demonstrated that at dose 100 and 50mg/kg B.W. (P<0.01) and 25mg/kg B.W. (P<0.05) of GPA pretreated dramatically prevented ANIT induced decreased in bile flow rate. Compared with ANIT treated group, the results of bile biochemical parameters about total bile acid (TBA) was increased by GPA at groups with any dose (P<0.01), glutathione (GSH) was increased significantly at high dose (P<0.01) and medium dose (P<0.05), total bilirubin (TB) was increased at high and medium dose (P<0.05), direct bilirubin (DB) was only increased at high dose (P<0.01). Serum levels of glutamic-Oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), γ-glutamyltranspeptidase (γ-GT), TB, DB and TBA in comparison with ANIT treated group (P<0.01) were reduced by GPA (between 100 and 50mg/kg B.W.) pretreatment. Histopathology of the liver tissue showed that pathological damages and hepatic portal area filled with bile were relieved after GPA pretreatment compared with ANIT treated group. The protein and mRNA expression of Fxr, Bsep and Mrp2 were decreased in ANIT treated group. On the contrary, the protein and mRNA of Fxr, Bsep and Mrp2 were up regulated significantly pretreatment by GPA at dose of high and medium groups. On protein level of Bsep and Mrp2 the result shown no statistical difference in GPA (25mg/kg B.W.), but it was not same shown in mRNA level. CONCLUSION: The results of this investigation have demonstrated that the GPA exerts a dose dependent hepatoprotection effect on ANIT induced liver damage with acute intrahepatic cholestasis in rats, which may due to Fxr mediated regulation of bile transporters like Bsep and Mrp2.

Modulation of Multidrug Resistance Protein 2 Efflux in the Cisplatin Resistance Human Ovarian Carcinoma Cells A2780/RCIS by Sesquiterpene Coumarins.

Recent in vitro studies showed that sesquiterpene coumarins (SCs) can be used as chemosensitizers. In this study, 14 SCs were isolated and purified from roots of four Ferula species and their structures were elucidated by spectroscopic methods. The purified SCs were evaluated for multidrug resistance (MDR) reversal properties in A2780/RCIS cells (cisplatin-resistant derivatives of the human ovarian carcinoma cell line A2780P). Among the tested compounds, mogoltacin, mogoltadone, farnesiferol A, farnesiferol B, farnesiferol C, lehmferin, conferdione, and samarcandin showed significant MDR reversing effects. The combination of nontoxic concentrations of SCs (20 µM) with cisplatin enhanced cisplatin cytotoxicity on A2780/RCIS cells significantly. Flow cytometric efflux assay confirmed that the intracellular accumulation of 5-carboxyfluorescein diacetate (5-CFDA) was significantly increased in A2780/RCIS cells when treated with SCs. Our findings revealed that conferdione and samarcandin possessed the highest inhibitory effects on multidrug resistance-associated protein 2 pump efflux, and therefore, these compounds could be considered as lead scaffolds for further structure modifications.

Tissue distribution and ontogeny of multidrug resistance protein 2, a phosphatidylcholine translocator, in rats.

Multidrug resistance protein 2 (Mdr2), encoded by ATP-binding cassette b4 (Abcb4), serves as a phospholipid flippase that is indispensable for phosphatidylcholine translocation. However, little was known about the regulation of Mdr2 in Sprague-Dawley rats, although they are commonly used for pre-clinical investigation as well as mechanistic study. Present study aims at determining the tissue distribution, gender difference and ontogeny of Mdr2 in rats on both gene and protein levels. Results showed that Mdr2 was highly expressed in liver, modestly enriched in brain and testis, and less distributed in gastrointestinal tracts. Gender-divergent and male-dominated distribution was observed in the Mdr2 mRNA expression of liver and generative organs. Developmental pattern of rat Mdr2 on protein level was not exactly consistent with that on mRNA level. In conclusion, there was a considerable distribution of rat Mdr2 in the brain, testis and intestine besides liver, and the ontogeny of Mdr2 performed in an age-dependent pattern with the post-transcriptional regulation.

MK571 inhibits phase-2 conjugation of flavonols by Caco-2/TC7 cells, but does not specifically inhibit their apical efflux.

MK571 is a multidrug resistance protein-2 (ABCC2, Mrp2) inhibitor and has been widely used to demonstrate the role of Mrp2 in the cellular efflux of drugs, xenobiotics and their conjugates. Numerous reports have described modulation of Caco-2 cellular efflux and transport of flavonoids in the presence of MK571. Since flavonoids are efficiently conjugated by Caco-2/TC7 cells, we investigated the effects of MK571 on the efflux of flavonoid conjugates. The flavonol aglycones kaempferol, quercetin and galangin were efficiently taken up, conjugated and effluxed by Caco-2/TC7 cells. Apically-applied MK571 caused significant reductions in both the apical and basolateral efflux of flavonol conjugates from Caco-2/TC7 monolayers. MK571 did not significantly alter the apical:basolateral efflux ratio for flavonol conjugates, however, which is not consistent with MK571 specifically inhibiting only apical Mrp2. Since MK571 decreased the total amounts of conjugates formed, and increased cellular flavonol aglycone concentrations, we explored the possibility that MK571 also inhibits phase-2 conjugation of flavonols. MK571 dose-dependently inhibited the intracellular biosynthesis of all flavonol glucuronides and sulphates by Caco-2 cells. MK571 significantly inhibited phase-2 conjugation of kaempferol by cell-free extracts of Caco-2, and production of kaempferol-4'-O-glucuronide was competitively inhibited. These data show that MK571, in addition to inhibiting MRP2, is a potential inhibitor of enterocyte phase-2 conjugation.

Sensitivity and resistance to treatment in the primary management of epithelial ovarian cancer.

Ovarian carcinoma is the most lethal gynaecologic malignancy. Despite wide initial sensibility to chemotherapy especially to platinum-based regimens, the vast majority of patients with advanced stages of the disease develop recurrences and subsequent resistance to treatments. Ovarian cancer is actually considered as a heterogeneous disease at the clinical, histological and molecular level. In this review, the mechanisms of intrinsic sensitivity or resistance to treatment, especially to platinum-based chemotherapy are considered with particular reference to the significance of tumour heterogeneity. The molecular features involved in acquired resistance are reviewed and the current hypotheses are discussed. In particular, potential disruptions of the DNA reparation pathways are highlighted.

Resveratrol attenuates cisplatin renal cortical cytotoxicity by modifying oxidative stress.

Cisplatin, a cancer chemotherapy drug, is nephrotoxic. The aim of this study was to investigate whether resveratrol (RES) reduced cisplatin cytotoxicity and oxidative stress. Rat renal cortical slices were pre-incubated 30min with 0 (VEH, ethanol) or 30µg/ml RES followed by 60, 90 or 120min co-incubation with 0, 75, or 150µg/ml cisplatin. Lactate dehydrogenase (LDH) leakage was unchanged at 60 and 90min by cisplatin. Cisplatin increased (p<0.05) LDH leakage at 120min which was protected by RES. Cisplatin induced oxidative stress prior to LDH leakage as cisplatin depressed glutathione peroxidase and superoxide dismutase (SOD) activity, increased lipid peroxidation, protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins within 60min. RES failed to reverse glutathione (GSH) depression by cisplatin. In order to eliminated an extracellular interaction between RES and cisplatin, additional studies (RINSE studies) allowed a 30min RES uptake into slices, transfer of slices to buffer lacking RES, followed by 120min cisplatin incubation. RES in the RINSE studies prevented LDH leakage by cisplatin indicating that RES protection was not via a physical interaction with cisplatin in the media. These findings indicate that RES diminished cisplatin in vitro renal toxicity and prevented the development of oxidative stress.

Activation of human biliverdin-IXα reductase by urea: generation of kinetically distinct forms during the unfolding pathway.

Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3M urea, the Vmax is increased 11-fold to 1.8µmol/min/mg and the apparent Km for biliverdin increases from 1 to 3µM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent Vmax was shown to decay to half that seen at zero time with a half life of 5.8minutes, while the apparent Km for NADPH remains constant at approximately 5µM. With NADH as cofactor the half life of the activated (A) form was 2.9minutes, and this form decays in 3M urea to a less active (LA) form. The apparent Km for NADH increases from 0.33mM to 2mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells.

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.

Comparison of culture media for bile Acid transport studies in primary human hepatocytes.

BACKGROUND: Primary human hepatocytes are a useful in vitro model system to examine hepatic biochemical pathways, liver disorders and/or pharmacotherapies. This system can also be used for transport studies to investigate uptake and excretion of bile acids. Proper modeling of hepatic function requires careful attention to media components, and culture substrates and conditions. OBJECTIVES: To examine the effects of different culture media and conditions on bile acid transport in cultured human hepatocytes. METHODS AND RESULTS: Hepatocytes cultured in Williams' medium E showed an increase in both uptake and excretion of taurocholate compared to cells cultured in Dulbecco's Modified Eagle Medium (DMEM). Supplementation of DMEM with glutathione or ascorbic acid did not compensate for the lower transport. The difference can be explained by lower mRNA expression of the transporter proteins sodium taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP; ABCB11) when cultured in DMEM. Hepatocytes cultured in DMEM also display fewer and smaller bile canaliculi. Following extended time in culture supplementation of Williams' medium E with dexamethasone increased the expression of NTCP and BSEP. CONCLUSION: Williams' medium E is superior to DMEM for transport studies in primary human hepatocytes. Supplementation with dexamethasone increase mRNA levels of NTCP and BSEP.