Hyaluronan synthesis inhibition normalizes ethanol-enhanced hepatic stellate cell activation.

BACKGROUND: Chronic ethanol overconsumption promotes alcohol-associated liver disease (ALD), characterized by hepatocyte injury, inflammation, hepatic stellate cell (HSC) activation, and fibrosis. Hyaluronan (HA) concentration is greater in livers and blood from advanced ALD patients than patients with advanced non-ALD. In the liver, HSCs are the major HA producers. The relationship between ethanol, HA, and HSC activation is incompletely understood. Thus, here, we tested the hypothesis that ethanol enhances HSC activation in a HA-dependent manner. METHODS: Liver tissue microarrays (TMAs) containing steatotic livers from donors with or without a history of alcohol consumption were used to measure HA and collagen content. Mice were fed a moderate (2%, v/v) ethanol-containing diet or pair-fed control diet for 2 days, after which they were given a single carbon tetrachloride (CCl4 ) injection. To inhibit HA synthesis, we provided 4-methylumbelliferone (4MU) daily. We used LX2 cells, a human HSC cell line, to determine the impact ethanol had on LPS responses, with or without concurrent 4MU exposure. RESULTS: CCl4 induced liver injury, but it did not differ between ethanol or control diet fed mice with or without 4MU treatment. Ethanol feeding enhanced CCl4 -induced hepatic HA content, which was paralleled by HA synthase (Has)2 transcript abundance; 4MU treatment normalized both. Consistently, HSC activation, assessed by measuring αSMA mRNA and protein, was induced by CCl4 exposure, enhanced by ethanol feeding, and normalized by 4MU. Hepatic transcripts, but not protein, for Ccl2 were enhanced by ethanol feeding and normalized by 4MU exposure. Finally, ethanol-exposed LX2 cells made more LPS-stimulated CCL2 mRNA and protein than cells not exposed to ethanol; 4MU prevented this. CONCLUSION: These data show that ethanol augments HSC activation through HA synthesis and enhances hepatic profibrogenic features. Therefore, targeting HSC HA production could potentially attenuate liver disease in ALD patients.

In vitro evaluation suggests fenfluramine and norfenfluramine are unlikely to act as perpetrators of drug interactions.

Studies support the safety and efficacy of fenfluramine (FFA) as an antiseizure medication (ASM) in Dravet syndrome, Lennox-Gastaut syndrome, or CDKL5 deficiency disorder, all pharmacoresistant developmental and epileptic encephalopathies. However, drug-drug interactions with FFA in multi-ASM regimens have not been fully investigated. We characterized the perpetrator potential of FFA and its active metabolite, norfenfluramine (nFFA), in vitro by assessing cytochrome P450 (CYP450) inhibition in human liver microsomes, CYP450 induction in cultured human hepatocytes, and drug transporter inhibition potential in permeability or cellular uptake assays. Mean plasma unbound fraction was ~50% for both FFA and nFFA, with no apparent concentration dependence. FFA and nFFA were direct in vitro inhibitors of CYP2D6 (IC50 , 4.7 and 16 µM, respectively) but did not substantially inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, or CYP3A4/5. No time- or metabolism-dependent CYP450 inhibition occurred. FFA and nFFA did not induce CYP1A2; both induced CYP2B6 (up to 2.8-fold and up to 2.0-fold, respectively) and CYP3A4 (1.9- to 3.0-fold and 3.6- to 4.8-fold, respectively). Mechanistic static pharmacokinetic models predicted that neither CYP450 inhibition nor induction was likely to be clinically relevant at doses typically used for seizure reduction (ratio of area under curve [AUCR] for inhibition <1.25; AUCR for induction >0.8). Transporters OCT2 and MATE1 were inhibited by FFA (IC50 , 19.8 and 9.0 µM) and nFFA (IC50 , 5.2 and 4.6 µM) at concentrations higher than clinically achievable; remaining transporters were not inhibited. Results suggest that FFA and nFFA are unlikely drug-drug interaction perpetrators at clinically relevant doses of FFA (0.2-0.7 mg/kg/day).

In vitro evaluation of fenfluramine and norfenfluramine as victims of drug interactions.

Fenfluramine (FFA) has potent antiseizure activity in severe, pharmacoresistant childhood-onset developmental and epileptic encephalopathies (e.g., Dravet syndrome). To assess risk of drug interaction affecting pharmacokinetics of FFA and its major metabolite, norfenfluramine (nFFA), we conducted in vitro metabolite characterization, reaction phenotyping, and drug transporter-mediated cellular uptake studies. FFA showed low in vitro clearance in human liver S9 fractions and in intestinal S9 fractions in all three species tested (t1/2  > 120 min). Two metabolites (nFFA and an N-oxide or a hydroxylamine) were detected in human liver microsomes versus six in dog and seven in rat liver microsomes; no metabolite was unique to humans. Selective CYP inhibitor studies showed FFA metabolism partially inhibited by quinidine (CYP2D6, 48%), phencyclidine (CYP2B6, 42%), and furafylline (CYP1A2, 32%) and, to a lesser extent (<15%), by tienilic acid (CYP2C9), esomeprazole (CYP2C19), and troleandomycin (CYP3A4/5). Incubation of nFFA with rCYP1A2, rCYP2B6, rCYP2C19, and rCYP2D6 resulted in 10%-20% metabolism and no clear inhibition of nFFA metabolism by any CYP-selective inhibitor. Reaction phenotyping showed metabolism of FFA by recombinant human cytochrome P450 (rCYP) enzymes rCYP2B6 (10%-21% disappearance for 1 and 10 µM FFA, respectively), rCYP1A2 (22%-23%), rCYP2C19 (49%-50%), and rCYP2D6 (59%-97%). Neither FFA nor nFFA was a drug transporter substrate. Results show FFA metabolism to nFFA occurs through multiple pathways of elimination. FFA dose adjustments may be needed when administered with strong inhibitors or inducers of multiple enzymes involved in FFA metabolism (e.g., stiripentol).

An FGF15/19-TFEB regulatory loop controls hepatic cholesterol and bile acid homeostasis.

Bile acid synthesis plays a key role in regulating whole body cholesterol homeostasis. Transcriptional factor EB (TFEB) is a nutrient and stress-sensing transcriptional factor that promotes lysosomal biogenesis. Here we report a role of TFEB in regulating hepatic bile acid synthesis. We show that TFEB induces cholesterol 7α-hydroxylase (CYP7A1) in human hepatocytes and mouse livers and prevents hepatic cholesterol accumulation and hypercholesterolemia in Western diet-fed mice. Furthermore, we find that cholesterol-induced lysosomal stress feed-forward activates TFEB via promoting TFEB nuclear translocation, while bile acid-induced fibroblast growth factor 19 (FGF19), acting via mTOR/ERK signaling and TFEB phosphorylation, feedback inhibits TFEB nuclear translocation in hepatocytes. Consistently, blocking intestinal bile acid uptake by an apical sodium-bile acid transporter (ASBT) inhibitor decreases ileal FGF15, enhances hepatic TFEB nuclear localization and improves cholesterol homeostasis in Western diet-fed mice. This study has identified a TFEB-mediated gut-liver signaling axis that regulates hepatic cholesterol and bile acid homeostasis.

Effects of monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and interferon-α2a on P450 enzymes in human hepatocytes in vitro.

Some immunomodulatory agents stimulate the release of cytokines capable of suppressing P450 enzymes and potentially affecting pharmacokinetics of coadministered medications. Cytokines released in response to an immunomodulator in the blood ex vivo can be used to screen for the potential for drug-drug interactions. Tilsotolimod, an investigational agonist of Toll-like receptor 9, stimulated the release of macrophage chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and interferon-α2a (INF-α2a) in blood obtained from healthy donors. Although tilsotolimod did not directly affect CYP1A2, CYP2B6, or CYP3A4 expression or activity, the cytokines stimulated by the drug reduced CYP1A2 and CYP2B6 enzyme activities in cultured human hepatocytes. This study sought to identify which cytokines were responsible for tilsotolimod's indirect effects on P450 enzymes in vitro. A 72-h treatment with recombinant human chemokines MCP-1 and MIP-1α did not alter CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP3A4, or signal transducer and activator of transcription 1 (STAT1) mRNA expression or CYP1A2, CYP2B6, or CYP3A4/5 enzyme activity in cocultures of human hepatocytes and Kupffer cells. INF-α2a, at 2.5 ng/mL but not at the lower concentrations applied to the cells, increased CYP1A2 and STAT1 mRNA by 2.4- and 5.2-fold, respectively, and reduced CYP2B6 enzyme activity to 46% of control. This study established that INF-α2a, but not MCP-1 or MIP-1α, mediated tilsotolimod effects on CYP1A2 and CYP2B6 expression in human hepatocytes.

Direct and cytokine-mediated effects of albumin-fused growth hormone, TV-1106, on CYP enzyme expression in human hepatocytes in vitro.

Some biologics can modulate cytokines that may lead to changes in expression of drug-metabolizing enzymes and cause drug-drug interactions (DDI). DDI potential of TV-1106-an albumin-fused growth hormone (GH)-was investigated. In this study, human blood was exposed to recombinant human growth hormone (rhGH) or TV-1106, followed by isolation of the plasma and its application to human hepatocytes. While the treatment of blood with rhGH increased multiple cytokines, treatment of blood with TV-1106 had no effect on any of the nine cytokines tested. The interleukin (IL)-6 concentration was higher in the rhGH then in the TV-1106-treated plasma (P < .05). While rhGH had little or no effect on CYP1A2 or CYP2C19 mRNA but increased CYP3A4 mRNA twofold, TV-1106 had little or no effect on cytochrome P450 (CYP) mRNAs in hepatocytes. Although the plasma from rhGH-treated blood lowered CYP1A2 activity, the TV-1106 plasma had no effect on CYP activities. The CYP1A2 activity was lower in the rhGH- then in the TV-1106-plasma treated hepatocytes (P < .05). The results indicated that fusing GH with albumin made TV-1106 an unlikely participant of CYP1A2, CYP2C19 or CYP3A4-facilitated, direct or cytokine-driven DDI.

Membrane Assays to Characterize Interaction of Drugs with ABCB1.

ATP-binding cassette sub-family B member 1 (ABCB1) [P-glycoprotein (P-gp), multidrug resistance protein 1 (MDR1)] can affect the pharmacokinetics, safety, and efficacy of drugs making it important to identify compounds that interact with ABCB1. The ATPase assay and vesicular transport (VT) assay are membrane based assays that can be used to measure the interaction of compounds with ABCB1 at a lower cost and higher throughput compared to cellular-based assays and therefore can be used earlier in the drug development process. To that end, we tested compounds previously identified as ABCB1 substrates and inhibitors for interaction with ABCB1 using the ATPase and VT assays. All compounds tested interacted with ABCB1 in both the ATPase and VT assays. All compounds previously identified as ABCB1 substrates activated ABCB1-mediated ATPase activity in the ATPase assay. All compounds previously identified as ABCB1 inhibitors inhibited the ABCB1-mediated transport in the VT assay. Interestingly, six of the ten compounds previously identified as ABCB1 inhibitors activated the basal ATPase activity in activation assays suggesting that the compounds are substrates of ABCB1 but can inhibit ABCB1 in inhibition assays. Importantly, for ATPase activators the EC50 of activation correlated with the IC50 values from the VT assay showing that interactions of compounds with ABCB1 can be measured with similar levels of potency in either assay. For ATPase nonactivators the IC50 values from the ATPase inhibition and VT inhibition assay showed correlation. These results demonstrate the utility of membrane assays as tools to detect and rank order drug-transporter interactions.

Anti-CD28 monoclonal antibody-stimulated cytokines released from blood suppress CYP1A2, CYP2B6, and CYP3A4 in human hepatocytes in vitro.

Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood-mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism-namely, the release of cytokines from PBMCs.

Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

Overexpression of glutathione-S-transferase, MGSTII, confers resistance to busulfan and melphalan.

A major obstacle to hematopoietic gene therapy is the lack of appropriate in vivo selection protocols that can raise the presently low numbers of gene-altered stem cells to therapeutically useful levels. Overexpression of glutathione-S-transferases (GST), in combination with busulfan treatment, may provide an exploitable selection mechanism for hematopoietic gene therapy strategies. GST provides a major route of detoxification of a variety of xenobiotics, including alkylating agents used for myeloablative chemotherapy. The only known route of clearance of busulfan is by GST-mediated conjugation. Using a fibroblast cell line as a model, we have tested the effects of overexpression of three human GST (GSTA1, GSTP1, and MGSTII) on cell survival under a busulfan or melphalan challenge. In two separate assay formats using chronic exposure to busulfan, MGSTII conferred a reproducible twofold selective advantage. GSTA1 and GSTP1 had no effect on busulfan resistance, and melphalan resistance was not affected by expression of any of the GSTs in these assays. In an acute (24-hour) melphalan exposure assay, MGSTII conferred about a twofold selective advantage. Busulfan was not toxic in this assay. RTPCR analysis of human bone marrow CD34+ cells showed that MGSTII is not highly expressed in this stem/early progenitor population. These data indicate that MGSTII may be a useful selective agent in hematopoietic gene therapy.

Effects of prototypical microsomal enzyme inducers on cytochrome P450 expression in cultured human hepatocytes.

Cultured human hepatocytes are a valuable in vitro system for evaluating new molecular entities as inducers of cytochrome P450 (P450) enzymes. The present study summarizes data obtained from 62 preparations of cultured human hepatocytes that were treated with vehicles (saline or dimethylsulfoxide, 0.1%), beta-naphthoflavone (33 microM), phenobarbital (100 or 250 microM), isoniazid (100 microM) and/or rifampin (20 or 50 microM), and examined for the expression of P450 enzymes based on microsomal activity toward marker substrates, or in the case of CYP2C8, the level of immunoreactive protein. The results show that CYP1A2 activity was markedly induced by beta-naphthoflavone (on average 13-fold, n = 28 preparations), and weakly induced by phenobarbital (1.9-fold, n = 25) and rifampin (2.3-fold, n = 22); CYP2A6 activity tended to be increased with phenobarbital (n = 7) and rifampin (n = 3) treatments, but the effects were not statistically significant; CYP2B6 was induced by phenobarbital (6.5-fold, n = 13) and rifampin (13-fold, n = 14); CYP2C8 was induced by phenobarbital (4.0-fold, n = 4) and rifampin (5.2-fold, n = 4); CYP2C9 was induced by phenobarbital (1.8-fold, n = 14) and rifampin (3.5-fold, n = 10); CYP2C19 was markedly induced by rifampin (37-fold, n = 10), but relatively modestly by phenobarbital (7-fold, n = 9); CYP2D6 was not significantly induced by phenobarbital (n = 5) or rifampin (n = 5); CYP2E1 was induced by phenobarbital (1.7-fold, n = 5), rifampin (2.2-fold, n = 5), and isoniazid (2.3-fold, n = 5); and, CYP3A4 was induced by phenobarbital (3.3-fold, n = 42) and rifampin (10-fold, n = 61), but not by beta-naphthoflavone. Based on these observations, we generalize that beta-naphthoflavone induces CYP1A2 and isoniazid induces CYP2E1, whereas rifampin and, to a lesser extent phenobarbital, tend to significantly and consistently induce enzymes of the CYP2A, CYP2B, CYP2C, CYP2E, and CYP3A subfamilies but not the 2D subfamily.