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).

Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice.

Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin+imipenem and cephalothin+neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin+imipenem but stimulated by cephalothin+neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism.

Dysfunction of organic anion transporting polypeptide 1a1 alters intestinal bacteria and bile acid metabolism in mice.

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in liver and is able to transport bile acids (BAs) in vitro. Male Oatp1a1-null mice have increased concentrations of taurodeoxycholic acid (TDCA), a secondary BA generated by intestinal bacteria, in both serum and livers. Therefore, in the present study, BA concentrations and intestinal bacteria in wild-type (WT) and Oatp1a1-null mice were quantified to investigate whether the increase of secondary BAs in Oatp1a1-null mice is due to alterations in intestinal bacteria. The data demonstrate that Oatp1a1-null mice : (1) have similar bile flow and BA concentrations in bile as WT mice; (2) have a markedly different BA composition in the intestinal contents, with a decrease in conjugated BAs and an increase in unconjugated BAs; (3) have BAs in the feces that are more deconjugated, desulfated, 7-dehydroxylated, 3-epimerized, and oxidized, but less 7-epimerized; (4) have 10-fold more bacteria in the small intestine, and 2-fold more bacteria in the large intestine which is majorly due to a 200% increase in Bacteroides and a 30% reduction in Firmicutes; and (5) have a different urinary excretion of bacteria-related metabolites than WT mice. In conclusion, the present study for the first time established that lack of a liver transporter (Oatp1a1) markedly alters the intestinal environment in mice, namely the bacteria composition.

Expression of hepatocytic- and biliary-specific transcription factors in regenerating bile ducts during hepatocyte-to-biliary epithelial cell transdifferentiation.

BACKGROUND: Under compromised biliary regeneration, transdifferentiation of hepatocytes into biliary epithelial cells (BEC) has been previously observed in rats, upon exposure to BEC-specific toxicant methylene dianiline (DAPM) followed by bile duct ligation (BDL), and in patients with chronic biliary liver disease. However, mechanisms promoting such transdifferentiation are not fully understood. In the present study, acquisition of biliary specific transcription factors by hepatocytes leading to reprogramming of BEC-specific cellular profile was investigated as a potential mechanism of transdifferentiation in two different models of compromised biliary regeneration in rats. RESULTS: In addition to previously examined DAPM + BDL model, an experimental model resembling chronic biliary damage was established by repeated administration of DAPM. Hepatocyte to BEC transdifferentiation was tracked using dipetidyl dipeptidase IV (DDPIV) chimeric rats that normally carry DPPIV only in hepatocytes. Following DAPM treatment, ~20% BEC population turned DPPIV-positive, indicating that they are derived from DPPIV-positive hepatocytes. New ductules emerging after DAPM + BDL and repeated DAPM exposure expressed hepatocyte-associated transcription factor hepatocyte nuclear factor (HNF) 4α and biliary specific transcription factor HNF1ß. In addition, periportal hepatocytes expressed biliary marker CK19 suggesting periportal hepatocytes as a potential source of transdifferentiating cells. Although TGFß1 was induced, there was no considerable reduction in periportal HNF6 expression, as observed during embryonic biliary development. CONCLUSIONS: Taken together, these findings indicate that gradual loss of HNF4α and acquisition of HNF1ß by hepatocytes, as well as increase in TGFß1 expression in periportal region, appear to be the underlying mechanisms of hepatocyte-to-BEC transdifferentiation.

Expression of specific hepatocyte and cholangiocyte transcription factors in human liver disease and embryonic development.

Transcription factors are major determinants of cell-specific gene expression in all cell types. Studies in rodent liver have shown that alterations in transcription factor expression determine lineage specification during fetal liver development and signify transdifferentiation of cells of the biliary compartment into 'oval' cells and eventually hepatocytes in adult liver. We examined the cellular localization of hepatocyte- or BEC-associated transcription factors in human fetal and adult liver and in diseases in which transdifferentiation between hepatocytes and biliary cells may play a role. In the normal adult human liver, hepatocyte nuclear factor (HNF)4 alpha and HNF6 appeared exclusively in hepatocytes; HNF1beta, HNF3alpha, and HNF3beta were observed only in BEC. During fetal development both BEC and hepatocytes expressed HNF3alpha, HNF3beta, and HNF6. HNF1alpha was expressed only in fetal hepatocytes. We further examined expression of transcription factors in massive hepatic necrosis and in specific types of chronic liver disease. Hepatocyte-associated transcription factors HNF4 alpha and HNF6 also appeared in BEC in massive hepatic necrosis and chronic hepatitis C virus infection. Similarly, HNF3beta that is expressed only in BEC in normal adult liver was also observed in hepatocytes in primary biliary cirrhosis and chronic biliary obstruction. These data mimic previous findings in rodents in which hepatocyte-associated transcription factors appear in biliary cells prior to emergence of oval cells, which function as progenitor cells for hepatocytes when the regenerative capacity of the latter is compromised.

Mechanisms of hepatocyte growth factor-mediated and epidermal growth factor-mediated signaling in transdifferentiation of rat hepatocytes to biliary epithelium.

UNLABELLED: Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/beta-catenin pathway, transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF+EGF. Most of the HGF+EGF target genes were altered by LY294002. CONCLUSION: Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

Upregulation of calpastatin in regenerating and developing rat liver: role in resistance against hepatotoxicity.

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCl(4)-induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP-bioactivating enzymes and covalent binding of the APAP-derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered.

Calpain: a death protein that mediates progression of liver injury.

Calpain is a Ca(2+)-regulated cytosolic cysteine protease that exists mainly in two isoforms and mediates crucial cellular functions, including rearrangement of cytoskeletal proteins, transport of the glucose transporter GLUT4, and protein cleavage to activate various receptors and pro-enzymes. Unintentional activation or functional loss of intracellular calpain has been implicated in several pathologies, including neurodegenerative diseases, traumatic brain and spinal cord injuries, cataracts and ischemia-associated injuries. Furthermore, polymorphism in the gene encoding calpain-10 has been associated with increased risk of type 2 diabetes. Recent studies have revealed a novel role for calpain in the progression of toxicant-induced liver damage. Evidence suggests that calpain leaking out of necrotic hepatocytes is highly activated in the extracellular milieu and hydrolyzes proteins in the plasma membrane of neighboring cells leading to progression of injury. Experimental intervention with calpain inhibitors substantially mitigates progression of liver injury initiated by toxicants, thereby preventing acute liver failure, and toxicant-induced animal death, pointing to a new potential therapeutic strategy against acute toxicities.

Potentiation of carbon tetrachloride hepatotoxicity and lethality in type 2 diabetic rats.

There is a need for well characterized and economical type 2 diabetic model that mimics the human disease. We have developed a type 2 diabetes rat model that closely resembles the diabetic patients and takes only 24 days to develop robust diabetes. Nonlethal doses of allyl alcohol (35 mg/kg i.p.), CCl(4) (2 ml/kg i.p.), or thioacetamide (300 mg/kg i.p.) yielded 80 to 100% mortality in diabetic rats. The objective of the present study was to investigate two hypotheses: higher CCl(4) bioactivation and/or inhibited compensatory tissue repair were the underlying mechanisms for increased CCl(4) hepatotoxicity in diabetic rats. Diabetes was induced by feeding high fat diet followed by a single dose of streptozotocin on day 14 (45 mg/kg i.p.) and was confirmed on day 24 by hyperglycemia, normoinsulinemia, and oral glucose intolerance. Time course studies (0-96 h) of CCl(4) (2 ml/kg i.p.) indicated that although initial liver injury was the same in nondiabetic and diabetic rats, it progressed only in the latter, culminating in hepatic failure, and death. Hepatomicrosomal CYP2E1 protein and activity, lipid peroxidation, glutathione, and (14)CCl(4) covalent binding to liver tissue were the same in both groups, suggesting that higher bioactivation-based injury is not the mechanism. Inhibited tissue repair resulted in progression of injury and death in diabetic rats, whereas in the nondiabetic rats robust tissue repair resulted in regression of injury and survival after CCl(4) administration. These studies show high sensitivity of type 2 diabetes to model hepatotoxicants and suggest that CCl(4) hepatotoxicity is potentiated due to inhibited tissue repair.

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants.

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.

Mechanisms of increased liver tissue repair and survival in diet-restricted rats treated with equitoxic doses of thioacetamide.

Moderate dietary or caloric restriction (DR) modulates animal physiology in a beneficial fashion. Previously, we have reported an equitoxic dose experiment where liver injury in DR male Sprague-Dawley rats exposed to a low dose of thioacetamide (TA, 50 mg/kg) was similar to that observed in ad libitum fed (AL) rats exposed to a 12-fold higher dose (600 mg/kg). Paradoxically, the AL rats experienced 90% mortality while all of the DR rats, with the same amount of initial bioactivation-mediated liver injury, survived. The protection observed in the DR rats was due to efficient compensatory liver tissue repair, which was delayed and attenuated in the AL rats, leading to progression of liver injury. The objective of the present study was to investigate the molecular mechanisms of the enhanced tissue repair in the DR rats upon equitoxic challenge with TA. Promitogenic mechanisms and mediators such as proinflammatory cytokines (TNF-alpha and IL-6), growth factors (TGF-alpha and HGF), and inducible nitric oxide synthase (iNOS) were estimated over a time course after equitoxic challenge (50 mg/kg to DR vs. 600 mg/kg to AL rats). Except for TNF-alpha, all other molecules were expressed earlier and in greater amount in the DR rats. IL-6 was 10-fold greater and peaked 12 h earlier; HGF also peaked 12 h sooner in the DR rats, when it was 2.5-fold greater than the value in the AL rats. TGF-alpha expression in livers of DR rats increased after TA administration and peaked at 24 h. In the AL rats, it was lower and peaked at 36 h. Diet restriction alone induced iNOS 2-fold in the DR rats and remained elevated until 12 h after TA administration, then declined thereafter. The lower iNOS activity in the AL rats further decreased after TA injection. DR rats exhibited higher apoptosis after thioacetamide administration, which further increased the efficiency of tissue repair. Taken together, these data indicate that even though the liver injury is near equal in AL and DR rats, sluggish signal transduction leads to delayed liver regeneration, progression of liver injury, and death in the AL rats. The equitoxic dose experiment indicates that stimulation of tissue repair is independent of the extent of initial liver injury and is governed by physiology of diet restriction. DR stimulates promitogenic signaling leading to a quick and timely response upon liver injury, arrest of progressive injury on one hand, and recovery from injury on the other, paving the way for survival of the DR rats.

Upregulated promitogenic signaling via cytokines and growth factors: potential mechanism of robust liver tissue repair in calorie-restricted rats upon toxic challenge.

Previously we reported that moderate calorie restriction or diet restriction (DR, calories reduced by 35% for 21 days) in male Sprague-Dawley rats protects from a lethal dose of thioacetamide (TA). DR rats had 70% survival compared with 10% in rats fed ad libitum (AL) because of timely and adequate compensatory liver cell division and tissue repair in the DR rats. Further investigation of the mechanisms indicate that enhanced promitogenic signaling plays a critical role in this stimulated tissue repair. Expression of stimulators of promitogenic signaling interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), hepatocyte growth factor (HGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) were studied during liver tissue repair after TA-induced liver injury. Plasma IL-6 was significantly higher in the DR rats, with 6-fold higher expression at 48 h after TA administration. Immunohistochemical localization revealed significantly higher expression of IL-6 in the hepatic sinusoidal endothelium of DR rats. Expression of TGF-alpha and HGF was consistently higher in the livers of DR rats from 36 to 72 h. EGFR, which serves as a receptor for TGF-alpha, was higher in DR rats before TA administration and remained higher till 48 h after TA intoxication. DR-induced 2-fold increase in hepatic iNOS activity is consistent with early cell division in DR rats after TA challenge. These data suggest that the reason behind the higher liver tissue repair after TA-induced hepatotoxicity in DR rats is timely and higher expression of the growth stimulatory cytokines and growth factors. It appears that the physiological effects of DR make the liver cells vigilant and prime the liver tissue promptly for liver regeneration through promitogenic signaling upon toxic challenge.