Lithium Pre-Storage Enables High Initial Coulombic Efficiency and Stable Lithium-Enriched Silicon/Graphite Anode.

Application of silicon-based anodes is significantly challenged by low initial Coulombic efficiency (ICE) and poor cyclability. Traditional pre-lithiation reagents often pose safety concerns due to their unstable chemical nature. Achieving a balance between water-stability and high ICE in prelithiated silicon is a critical issue. Here, we present a lithium-enriched silicon/graphite material with an ultra-high ICE of ≥110 % through a high-stable lithium pre-storage methodology. Lithium pre-storage prepared a nano-drilled graphite material with surficial lithium functional groups, which can form chemical bonds with adjacent silicon during high-temperature sintering. This results in an unexpected O-Li-Si interaction, leading to in situ pre-lithiation of silicon nanoparticles and providing high stability in air and water. Additionally, the lithium-enriched silicon/graphite materials impart a combination of high ICE, high specific capacity (620 mAh g-1), and long cycling stability (>400 cycles). This study opens up a promising avenue for highly air- and water-stable silicon anode prelithiation methods.

CD47 and PD-L1 overexpression in proliferating human hepatocytes attenuated immune responses and ameliorated acute liver injury in mice.

Hepatocyte transplantation has the potential to treat acute liver failure and correct liver-based metabolic disorders. Proliferating human hepatocytes (ProliHHs) provide a large-scale source as an alternative to primary human hepatocytes. However, host rejection led to inefficient graft survival and function, which hindered the clinical application of cell therapy. Herein, we employed the lentiviral system to overexpress immunomodulatory factors programmed death-ligand 1 (cluster of differentiation 274) (CD274) and cluster of differentiation 47 (CD47) in ProliHHs. CD47+274 overexpression inhibited macrophage and T cell responses in vitro. After transplantation into mice via the spleen without immunosuppression, CD47+274 ProliHHs accumulation in the liver significantly increased for 48 hours compared with ProliHHs. Consistent with the in vitro results, CD47+274 ProliHHs were less aggregated and infiltrated by macrophages and also recruited fewer T cells in the liver. Seven days after transplantation, the human albumin level of engineered ProliHHs doubled compared with control group. CD47+274 ProliHHs further ameliorated the liver injury induced using concanavalin A. Overall, our results suggested CD47+274 overexpression reduced innate and adaptive immune responses during hepatocyte transplantation, and the survival rate and graft function of transplanted hepatocyte-like cells were all significantly improved.

MSCs cell fates in murine acute liver injury and chronic liver fibrosis induced by carbon tetrachloride.

Mesenchymal stem cells (MSCs) therapy has shown potential benefits in multiple diseases. However, their clinic performance is not as satisfactory as expected. This study aimed to provide an alternative explanation by comparing MSCs' fates in different liver diseases. The distribution and therapeutic effects of hMSCs were investigated in acute liver injury (ALI) and chronic liver fibrosis (CLF) mice models, respectively. The two models were induced by single or repeated injection of carbon tetrachloride (CCl4) separately. The increase of hMSCs exposure in the liver (AUCliver 0-72 h) were more significant in ALI than in CLF (177.1% vs. 96.2%). In the ALI model, the hMSCs exposures in the lung (AUClung 0-72 h) increased by nearly 50% while decreased by 60.7% in CLF. The efficacy satellite study indicated that hMSCs could significantly ameliorate liver injury in ALI, but its effects in CLF were limited. In the ALI, suppressed Natural Killer (NK) cell activities were observed, while NK cell activities were increased in CLF. The depletion of NK cells could increase hMSCs exposure in mice. For mice MSC (mMSCs), their cell fates in ALI were very similar to hMSCs in ALI: mMSCs' exposure in the liver and lung increased in ALI. In conclusion, our study revealed the distinct cell pharmacokinetic patterns of MSCs in ALI and CLF mice, which might be at least partially attributed to the different NK cell activities in the two liver diseases. This finding provided a novel insight into the varied MSCs' therapeutic efficacy in the clinic. Significance Statement Currently, there is little knowledge about the PK behavior of cell products like MSCs. This study was the first time investigating the influence of liver diseases on cell fates and efficacies of MSCs and the underneath rationale. The exposure was distinct between two representative liver disease models, which directly linked with the therapeutic performance that MSCs achieved. The difference could be attributed to the NK cells-mediated MSCs clearance.

Farnesoid X receptor (FXR) agonists induce hepatocellular apoptosis and impair hepatic functions via FXR/SHP pathway.

Farnesoid X receptor (FXR) plays an indispensable role in liver homeostasis and has been a promising drug target for hepatic diseases. However, the concerns of undesired biological actions limit the clinical applications of FXR agonists. To reveal the intrinsic mechanism of FXR agonist-induce hepatotoxicity, two typical FXR agonists with different structures (obeticholic acid (OCA) and Px-102) were investigated in the present study. By detecting MMP, ROS, and ATP and analyzing the fate of cells, we found that both OCA and Px-102 reduced the mitochondrial function of hepatocytes and promoted cell apoptosis. Gene ablation or inhibition of FXR or SHP ameliorated the cytotoxicities of OCA and Px-102, which indicated the adverse actions of FXR/SHP activation including down-regulation of phosphorylation of PI3K/AKT and functional hepatic genes. The dose-related injurious effects of OCA (10 mg/kg and 30 mg/kg) and Px-102 (5 mg/kg and 15 mg/kg) on the liver were confirmed on a high-fat diet mouse model. The decrease of hepatocyte-specific genes and augmenter of liver regeneration in the liver caused by OCA or Px-102 suggested an imbalance of liver regeneration and a disruption of hepatic functions. Exploration of intestinally biased FXR agonists or combination of FXR agonist with apoptosis inhibitor may be more beneficial strategies for liver diseases.

Define Mesenchymal Stem Cell from Its Fate: Biodisposition of Human Mesenchymal Stem Cells in Normal and Concanavalin A-Induced Liver Injury Mice.

The pharmaceutical industry and clinical trials have been revolutionized mesenchymal stem cell-based therapeutics. However, the pharmacokinetics of transplanted cells has been little characterized in their target tissues under healthy or disease condition. A quantitative polymerase chain reaction analytical method with matrix effect was developed to track the biodistribution of human mesenchymal stem cells in normal mice and those with Concanavalin A (Con A)-induced liver injury. Mesenchymal stem/stromal cell (MSC) disposition in blood and different organs were compared, and relevant pharmacokinetic parameters were calculated. Human MSCs (hMSCs) and mouse MSCs (mMSCs) displayed a very similar pharmacokinetic profile in all tested doses: about 95% of the detected hMSCs accumulated in the lung and 3% in the liver, and almost negligible cells were detected in other tissues. A significant double peak of hMSC concentration emerged in the lung within 1-2 hours after intravenous injection, as with mMSCs. Prazosin, a vasodilator, could eliminate the second peak in the lung and increase its Cmax and area under the concentration-time curve (AUC) by 10% in the first 2 hours. The injury caused by Con A was significantly reduced by hMSCs, and the Cmax and AUC0-8 (AUC from time 0 to 8 hours) of cells in the injured liver decreased by 54 and 50%, respectively. The Cmax and AUC would be improved with the alleviation of congestion through the administration of heparin. The study provides a novel insight into the pharmacokinetics of exogenous MSCs in normal and Con A-induced liver injury mice, which provides a framework for optimizing cell transplantation. SIGNIFICANCE STATEMENT: Mesenchymal stem/stromal cells (MSCs) are known for their potential as regenerative therapies in treating several diseases, but an insufficient understanding of the pharmacokinetics of MSCs restricts their future application. The current study was the first to elucidate the pharmacokinetics and possible factors, including dosage, species, and derived sources, in a systematic way. The study further revealed that Concanavalin A-induced liver injury significantly prevented cells from entering the injury site, which could be reversed by the diminished congestion achieved by heparin.

Functional Proliferating Human Hepatocytes: In Vitro Hepatocyte Model for Drug Metabolism, Excretion, and Toxicity.

To develop a functional alternative hepatocyte model for primary human hepatocytes (PHHs) with proliferative property, essential drug metabolic, and transporter functions, proliferating human hepatocytes (ProliHHs) expanded from PHHs were fully characterized in vitro. Herein, ProliHHs generated from multiple PHHs donors could be expanded more than 200-fold within four passages and maintained their metabolic or transporter capacities partially. Furthermore, ProliHHs were able to regain the mature hepatic property after three-dimensional (3D) culture. Particularly, the downregulated mRNA expression and function of three major cytochrome P450 (P450) enzymes (CYP1A2, CYP2B6, and CYP3A4) in the proliferating process (ProliHHs-P) could be recovered by 3D culture. The metabolic variabilities across different PHHs donors could be inherited to their matured ProliHHs (ProliHHs-M). The intrinsic clearances of seven major P450 enzymes in ProliHHs-M correlated well (r = 0.87) with those in PHHs. Also, bile canaliculi structures could be observed in sandwich-cultured ProliHHs (SC-ProliHHs), and the biliary excretion index of four probe compounds [cholyl-lys-fluorescein, 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate (CDF), deuterium-labeled sodium taurocholate acid, and rosuvastatin] in SC-ProliHHs (>10%) were close to sandwich-cultured PHHs. More importantly, both ProliHHs-P and ProliHHs-M could be used to evaluate hepatotoxicity. Therefore, these findings demonstrated that the 3D and sandwich culture system could be used to recover the metabolic and transporter functions in ProliHHs for clearance prediction and cholestasis risk assessment, respectively. Together, ProliHHs could be a promising substitute for PHHs in drug metabolism, transport, and hepatotoxicity screening. SIGNIFICANCE STATEMENT: This report describes the study of drug metabolic capacities, efflux transporter functions, and toxicity assessments of proliferating human hepatocytes (ProliHHs). The metabolic variability in different primary human hepatocyte donors could be inherited by their matured ProliHHs derivatives. Also, ProliHHs could form canalicular networks in sandwich culture and display biliary excretion capacities. More importantly, both the proliferative and maturation statuses of ProliHHs could be used to evaluate hepatotoxicity. Together, ProliHHs were feasible to support drug candidate screening in hepatic metabolism, disposition, and toxicity.

A facile method for vancomycin C-terminus functionalization and derivatization through hydrazide.

Over 60-year clinical use of vancomycin led to the emergence of vancomycin-resistant bacteria and threatened our health. To combat vancomycin-resistant strains, numerous vancomycin analogues were developed, such as Telavancin, Oritavancin and Dalbavancin. Extra structures embedded on C-terminus has been proved to be an effective strategy to promote antibacterial activity of vancomycin against vancomycin-resistant strains. Here, we reported a facile strategy, inspired by native chemical ligation, for vancomycin C-terminus functionalization and derivatization. The introduction of C-terminal hydrazide on vancomycin not only provided us an accessible method for C-terminus functionalization through carbonyl azide and thioester, also acted as an efficient site for vancomycin structure modifications. Based on hydrazide-vancomycin, we effectively conjugated cysteine and cysteine containing peptides onto vancomycin C-terminus, and two fluorescent FITC-vancomycin were prepared through Cys-Maleimide conjugation. Meanwhile, we introduced lipophilic structures onto vancomycin C-terminus via the hydrazide moiety. The obtained vancomycin derivatives were evaluated against both Gram-positive and negative bacteria strains.

Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-ßRI/Smad Signaling.

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-ß)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-ß receptor I (TGF-ßRI) and regulating the TGF-ß/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

A novel bile acid analog, A17, ameliorated non-alcoholic steatohepatitis in high-fat diet-fed hamsters.

Being endocrine signaling molecules that regulate lipid metabolism and affect energy balance, bile acids are potential drug candidates for non-alcoholic steatohepatitis (NASH). Obeticholic acid (OCA) could improve NASH accompanied by significant side effects. Therefore, it is worthwhile to develop safer and more effective bile acid analogs. In this study, a new bile acid analog A17 was synthesized and its potential anti-NASH effects were assessed in vitro and in vivo. The impact of A17 on steatosis was investigated in the rat primary hepatocytes challenged with oleic acid. It was found that A17 alleviated lipid accumulation by reducing fatty acid (FA) uptake and promoting FA oxidation. The reduction of FA uptake came from inhibiting fatty acid translocase (Cd36) expression. The promotion of FA oxidation came from stimulating the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha (AMPKα). In addition, A17 reduced lipopolysaccharide-induced inflammation in Raw264.7 cells by activating Takeda G protein-coupled receptor 5 (TGR5). In in vivo study, male Golden Syrian hamsters were fed with high fat (HF) diet and then treated with 50 mg/kg/d A17 for 6 weeks. A17 lowered the lipid profiles and liver enzyme levels in serum and improved liver pathological conditions with less side effects compared with OCA. Further studies confirmed that the molecular mechanisms of A17 in vivo were similar to those in vitro. In conclusion, a novel bile acid analog A17 was identified to ameliorate NASH in HF-fed hamsters. The potential mechanisms could be contributed to reducing FA uptake, stimulating FA oxidation and relieving inflammation.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF's active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5-100 µmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 µmol/L. TER (5 µmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells' uptake of acyclovir and increased the plasma concentration.

Dihydro-stilbene gigantol relieves CCl4-induced hepatic oxidative stress and inflammation in mice via inhibiting C5b-9 formation in the liver.

In general, anti-inflammatory treatment is considered for multiple liver diseases despite the etiology. But current drugs for alleviating liver inflammation have defects, making it necessary to develop more potent and safer drugs for liver injury. In this study, we screened a series of (dihydro-)stilbene or (dihydro-)phenanthrene derivatives extracted from Pholidota chinensis for their potential biological activities. Among 31 compounds, the dihydro-stilbene gigantol exerted most potent protective effects on human hepatocytes against lithocholic acid toxicity, and exhibited solid antioxidative and anti-inflammatory effect in vitro. In mice with CCl4-induced acute liver injury, pre-administration of gigantol (10, 20, 40 mg· kg-1· d-1, po, for 7 days) dose-dependently decreased serum transaminase levels and improved pathological changes in liver tissues. The elevated lipid peroxidation and inflammatory responses in the livers were also significantly alleviated by gigantol. The pharmacokinetic studies showed that gigantol was highly concentrated in the mouse livers, which consisted with its efficacy in preventing liver injury. Using a label-free quantitative proteomic analysis we revealed that gigantol mainly regulated the immune system process in liver tissues of CCl4-treated mice, and the complement and coagulation cascades was the predominant pathway; gigantol markedly inhibited the expression of complement component C9, which was a key component for the formation of terminal complement complex (TCC) C5b-9. These results were validated by immunohistochemistry (IHC) or real time-PCR. Confocal microscopy analysis showed that gigantol significantly inhibited the vascular deposition of TCC in the liver. In conclusion, we demonstrate for the first time that oral administration of gigantol potently relieves liver oxidative stress and inflammation, possibly via a novel mechanism of inhibiting the C5b-9 formation in the liver.

An Adaption of Human-Induced Hepatocytes to In Vitro Genetic Toxicity Tests.

Metabolic activation is essential in standard in vitro genotoxicity test systems. At present, there is a lack of suitable cell models that can express the major characteristics of liver function for predicting substance toxicity in humans. Human-induced hepatocytes (hiHeps), which have been generated from fibroblasts by lentiviral expression of liver transcription factors, can express hepatic gene programs and can be expanded in vitro and display functional characteristics of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Our purpose was to investigate whether hiHeps could be used as a more suitable model for genotoxicity evaluation of chemicals. Therefore, a direct mutagen, methylmethanesulfonate (MMS), and five promutagens [2-nitrofluorene (2-NF), benzo[a]pyrene (B[a]P), aflatoxin B1, cyclophosphamide and N-nitrosodiethylamine] were tested by the cytokinesis-block micronucleus test and the comet assay. Results from genotoxicity tests showed that the micronucleus frequencies were significantly increased by all of the six clastogens tested. Moreover, MMS, 2-NF and B[a]P induced significant increases in the % Tail DNA in the comet assay. In conclusion, our findings from the preliminary study demonstrated that hiHeps could detect the genotoxicity of indirect carcinogens, suggesting their potential to be applied as an effective tool for in vitro genotoxicity assessments.

Different structures of berberine and five other protoberberine alkaloids that affect P-glycoprotein-mediated efflux capacity.

Berberine, berberrubine, thalifendine, demethyleneberberine, jatrorrhizine, and columbamine are six natural protoberberine alkaloid (PA) compounds that display extensive pharmacological properties and share the same protoberberine molecular skeleton with only slight substitution differences. The oral delivery of most PAs is hindered by their poor bioavailability, which is largely caused by P-glycoprotein (P-gp)-mediated drug efflux. Meanwhile, P-gp undergoes large-scale conformational changes (from an inward-facing to an outward-facing state) when transporting substrates, and these changes might strongly affect the P-gp-binding specificity. To confirm whether these six compounds are substrates of P-gp, to investigate the differences in efflux capacity caused by their trivial structural differences and to reveal the key to increasing their binding affinity to P-gp, we conducted a series of in vivo, in vitro, and in silico assays. Here, we first confirmed that all six compounds were substrates of P-gp by comparing the drug concentrations in wild-type and P-gp-knockout mice in vivo. The efflux capacity (net efflux) ranked as berberrubine > berberine > columbamine ~ jatrorrhizine > thalifendine > demethyleneberberine based on in vitro transport studies in Caco-2 monolayers. Using molecular dynamics simulation and molecular docking techniques, we determined the transport pathways of the six compounds and their binding affinities to P-gp. The results suggested that at the early binding stage, different hydrophobic and electrostatic interactions collectively differentiate the binding affinities of the compounds to P-gp, whereas electrostatic interactions are the main determinant at the late release stage. In addition to hydrophobic interactions, hydrogen bonds play an important role in discriminating the binding affinities.

Roles of Hepatic Drug Transporters in Drug Disposition and Liver Toxicity.

Hepatic drug transporters are mainly distributed in parenchymal liver cells (hepatocytes), contributing to drug's liver disposition and elimination. According to their functions, hepatic transporters can be roughly divided into influx and efflux transporters, translocating specific molecules from blood into hepatic cytosol and mediating the excretion of drugs and metabolites from hepatic cytosol to blood or bile, respectively. The function of hepatic transport systems can be affected by interspecies differences and inter-individual variability (polymorphism). In addition, some drugs and disease can redistribute transporters from the cell surface to the intracellular compartments, leading to the changes in the expression and function of transporters. Hepatic drug transporters have been associated with the hepatic toxicity of drugs. Gene polymorphism of transporters and altered transporter expressions and functions due to diseases are found to be susceptible factors for drug-induced liver injury (DILI). In this chapter, the localization of hepatic drug transporters, their regulatory factors, physiological roles, and their roles in drug's liver disposition and DILI are reviewed.

Intestinal Transporter-Associated Drug Absorption and Toxicity.

Oral drug administration is the most favorable route of drug administration in the clinic. Intestinal transporters have been shown to play a significant role in the rate and extent of drug absorption of some, but not all, drug molecules. Due to the heterogeneous expression of multiple transporters along the intestine, the preferential absorption sites for drugs may vary significantly. In this chapter, we aim to summarize the current research on the expression, localization, function, and regulation of human intestinal transporters implicated in altering the absorption of low to medium molecular weight drug molecules. The role played by bile acid transport proteins (e.g., ASBT and OST-α/ß) is included in the discussion. The synergistic action of intestinal drug metabolism and transport is also discussed. Despite the complicated regulatory factors, the biopharmaceutics drug disposition classification system (BDDCS) put forward by Wu and Benet may help us better predict the effect of transporters on drug absorption. The drug-induced toxicity in the intestine, which may result from drug-drug interaction, gut microbiota, and bile salt toxicity, is also discussed.

Leflunomide Increases Hepatic Exposure to Methotrexate and Its Metabolite by Differentially Regulating Multidrug Resistance-Associated Protein Mrp2/3/4 Transporters via Peroxisome Proliferator-Activated Receptor α Activation.

Methotrexate (MTX) is the gold standard drug for the treatment of rheumatoid arthritis (RA), and it is frequently combined with leflunomide (LEF) to enhance its clinical efficacy. However, this combination can exacerbate liver toxicity, and the underlying mechanism has not yet been clarified. We investigated whether LEF affects the pharmacokinetics of MTX and its primary toxic metabolite, 7-hydroxyl methotrexate (7OH MTX), in mice. LEF significantly increased the plasma concentration (area under the plasma concentration-time curve) of MTX and 7OH MTX (2.4 and 4.5 times, respectively), decreased their bile excretion, and increased their accumulation in the liver and kidneys. When we investigated the effect of LEF on the MTX absorption, distribution, metabolism, and excretion process, we found that LEF had little effect on liver aldehyde oxidase and 7OH MTX formation. However, LEF significantly decreased the expression of the apical efflux transporter multidrug resistance-associated protein 2 (Mrp2) and increased that of the basolateral efflux transporters Mrp3/4, except there was no significant change in Mrp4 protein expression. Mrp2/3/4 alteration changed the distribution of MTX and 7OH MTX in plasma and tissues. Further studies suggested that LEF indirectly activated peroxisome proliferator-activated receptor α (PPARα), which was likely responsible for the Mrp2/3/4 alteration in the liver. The MTX plasma concentration change induced by LEF was reversed by the PPARα-specific antagonist GW6471. These results may partially explain the exacerbated liver toxicity caused by combination treatment with MTX and LEF and may raise concerns regarding the risk of potential drug-drug interactions between PPARα agonists and Mrp substrates in the clinic.

The involvement of multidrug and toxin extrusion protein 1 in the distribution and excretion of berberine.

1. Berberine (BBR), an isoquinoline alkaloid, has demonstrated multiple clinical pharmacological actions. As a substrate of multiple transporters in the liver, BBR is rarely excreted into the bile but can be found in the urine. The purpose of the present study was to investigate the role of multidrug and toxin extrusion protein 1 (MATE1) in the transport of BBR in the liver and kidney. 2. Using human MATE1 (hMATE1)-transfected HEK293 cells, BBR was shown to be a substrate of hMATE1 (Km = 4.28 ± 2.18 µM). In primary rat hepatocytes, pH-dependent uptake and efflux studies suggested that the transport of BBR was driven by the exchange of H+ and involved Mate1. In rats, we found that pyrimethamine (PYR), an inhibitor of Mate1, increased hepatic and renal distribution of BBR and decreased systematic excretion of BBR. 3. These findings indicated that BBR is a substrate of MATE1 and that hepatic and renal Mate1 promote excretion of BBR into bile and urine, respectively. In conclusion, Mate1 plays a key role in the distribution and excretion of BBR, and we speculate that drug-drug interactions (DDIs) caused by MATE1 may occur between BBR and other co-administered drugs.

Enhanced doxorubicin delivery to hepatocellular carcinoma cells via CD147 antibody-conjugated immunoliposomes.

HAb18G/CD147, an important marker in the progression of hepatocellular carcinoma (HCC), is highly expressed on the surface of HCC cells. To increase the therapeutic efficacy of Doxil (PEGylated liposomal doxorubicin) against HCC, we constructed CD147-targeted doxorubicin-loaded immunoliposomes (Anti-CD147 ILs-DOX) by conjugating F(ab')2 of a CD147-specific monoclonal antibody to DSPE-PEG-MAL, and then inserted the antibody-conjugated polymer to Doxil. Anti-CD147 ILs-DOX delivered DOX to CD147-overexpressing HCC cells specifically and efficiently in vitro and in vivo, resulting in enhanced therapeutic effects than non-targeted controls. Strikingly, Anti-CD147 ILs-DOX reduced the CD133-positive fraction of HCC cells, suggesting its potential in reducing the number of HCC stem cells. Pharmacokinetic and biodistribution studies of Anti-CD147 ILs-DOX confirmed its long circulation time and efficient accumulation in tumors. The superior antitumor effects of Anti-CD147 ILs-DOX than other treatments were demonstrated in both HCC cells and patient-derived HCC xenograft models. Anti-CD147 ILs-DOX represent a novel approach for targeted HCC therapy.

Hepatocellular carcinoma repression by TNFα-mediated synergistic lethal effect of mitosis defect-induced senescence and cell death sensitization.

UNLABELLED: Hepatocellular carcinoma (HCC) is a cancer lacking effective therapies. Several measures have been proposed to treat HCCs, such as senescence induction, mitotic inhibition, and cell death promotion. However, data from other cancers suggest that single use of these approaches may not be effective. Here, by genetic targeting of Survivin, an inhibitor of apoptosis protein (IAP) that plays dual roles in mitosis and cell survival, we identified a tumor necrosis factor alpha (TNFα)-mediated synergistic lethal effect between senescence and apoptosis sensitization in malignant HCCs. Survivin deficiency results in mitosis defect-associated senescence in HCC cells, which triggers local inflammation and increased TNFα. Survivin inactivation also sensitizes HCC cells to TNFα-triggered cell death, which leads to marked HCC regression. Based on these findings, we designed a combination treatment using mitosis inhibitor and proapoptosis compounds. This treatment recapitulates the therapeutic effect of Survivin deletion and effectively eliminates HCCs, thus representing a potential strategy for HCC therapy. CONCLUSION: Survivin ablation dramatically suppresses human and mouse HCCs by triggering senescence-associated TNFα and sensitizing HCC cells to TNFα-induced cell death. Combined use of mitotic inhibitor and second mitochondrial-derived activator of caspases mimetic can induce senescence-associated TNFα and enhance TNFα-induced cell death and synergistically eliminate HCC. (Hepatology 2016;64:1105-1120).

Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology.

BACKGROUND/AIMS: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. METHODS: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. RESULTS: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na+-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r2 = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. CONCLUSION: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.