Marcadores bioquímicos de ND en diabéticos tipo

mechanisms of hepatic uptake due to the technical ease of determining initial rates of uptake. To characterize uptake mechanism(s), various conditions have been applied in experiments using suspended hepatocytes, such as addition of inhibitor(s) for uptake pathways, substitution of sodium with choline, depletion of ATP and low temperature (Brock and Vore, 1984). One major disadvantage of suspended hepatocytes is that cells should be used within 6-8 h due to a rapid decrease in activity of both metabolic and transport proteins. In addition, during cell isolation,

hepatocytes lose polarity, and canalicular transport proteins such as P-gp and Mrp2 are internalized. Thus, use of suspended hepatocytes to determine biliary clearance is limited (Roelofsen et al., 1998; Bow et al., 2008).

3) Sandwich Cultured Hepatocytes: Hepatocytes cultured between two layers of gelled collagen in a sandwich configuration re-create the three-dimensional in vivo cellular environment and re-establish cell polarity forming canalicular membrane domains bounded by junctional complexes and bile canalicular networks (Liu et al., 1998; Liu et al., 1999c). In sandwich-cultured hepatocytes (SCH), transport proteins are expressed on the appropriate membrane domains (Hoffmaster et al., 2004a). For example, confocal microscopy revealed the co-expression of P-gp and Mrp2 on the canalicular domain, and biliary excretion of [D-penicillamine2,5]enkephalin (3H- DPDPE) and 5 (and 6)-carboxy-2',7'-dichlorofluorescein (CDF) confirmed P-gp and Mrp2 function, respectively. Expression and appropriate localization of transport proteins in SCH enables the utilization of this model to investigate drug-drug interactions mediated by inhibition of hepatobiliary excretion, and to elucidate the consequences of protein induction (Annaert et al., 2001; Zamek-Gliszczynski et al., 2003; Turncliff et al., 2004). SCH exhibit down-regulation of NTCP and up- regulation of MRP3; these changes may be due to the somewhat cholestatic nature of the canalicular networks that lack normal bile drainage via the common bile duct in vivo.

4) Isolated Perfused Liver Model: The isolated perfused rat liver system was first reported by Claude Bernard in 1855 and has been used to investigate the physiology and pathophysiology of the rat liver for many years (Wolkoff et al., 1979).

Although it is labor intensive and time- and animal-consuming, the isolated perfused liver system has been used widely to investigate hepatobiliary transport of drugs since it maintains intact hepatic architecture, remains viable for ~ 3 hr and allows for collection of perfusate and bile. This system is applicable for use with chemical modulators (e.g., inducers or inhibitors), and livers from rodents with transport protein(s) knocked out may be perfused to determine the contribution of specific proteins to hepatobiliary disposition. For example, in situ isolated perfused mouse livers have been used to identify the role of P-gp and Bcrp in the biliary excretion of 4-methylumbelliferyl sulfate (4-MUS) using P-gp- and Bcrp-deficient mice (Zamek- Gliszczynski et al., 2006a); the biliary excretion of 4-MUS was obliterated in Bcrp- deficient mice while 4-MUS biliary excretion was comparable between wild-type and P-gp-deficient mice, showing the exclusive role of Bcrp in 4-MUS biliary excretion. The use of 10 µM GF120918 in isolated perfused livers from wild-type mice showed significantly reduced biliary excretion of 4-MUS, which was comparable to that in livers from Bcrp-deficient mice.

5) Animal Knockout Models: Genetic models of transport protein deficiencies also have been used in in vivo pharmacokinetic and pharmacodynamic studies to assess the role of a particular transport protein in drug disposition. Animal models with transport protein deficiencies are a valuable tool to study hepatobiliary drug transport due to intact liver physiology and preserved hepatic metabolic activity and transport processes. However, the potential disadvantage of the knockout animal is the compensatory up-regulation of other metabolic enzymes and/or transport proteins as a result of the loss of a transport system. For example, the mutational disruption of

mdr1 genes led to over-expression of CYP3A, 2B, and 1A proteins and increased the catalytic activity of CYP enzymes (Schuetz et al., 2000). Interestingly, this up- regulation of CYP enzymes was observed only in P-gp deficient mice housed in Amsterdam, but not in P-gp deficient mice housed in the United States, suggesting that both the genetic deficiency and environmental factors regulated P450 expression in P-gp-deficient mice. Another example is the increased expression of basolateral Mrp3 in rodents with hereditary conjugated hyperbilirubinemia [Mrp2- deficient rats (GY/TR-) and Eisai hyperbilirubinemic rats (EHBR)] (Kuroda et al., 2004). This compensatory up-regulation of MRP3 also is observed in patients with Dubin-Johnson syndrome who suffer from impaired biliary excretion of organic anion due to the absence of MRP2 (Tsujii et al., 1999). Another limitation of using knockout animal models is the lack of predictability of these models to humans. For example, it has been documented previously that substrate specificity between the human MDR1 and mouse Mdr1a was markedly different, suggesting species differences in P-gp-mediated transport (Yamazaki et al., 2001). Species differences in the biliary excretion of phase II conjugates between rats and mice have been demonstrated recently (Zamek-Gliszczynski et al., 2008); Bcrp1 was involved exclusively in the biliary excretion of sulfate metabolites in mice, whereas both Mrp2 and Bcrp1 were responsible for canalicular transport of sulfate metabolites in rats.

Specific Aims

The goal of this dissertation project was to investigate the role of hepatic transport proteins in drug disposition and drug-induced liver injury. Transport proteins, individually and/or in concert, play a crucial role in hepatic uptake and excretion of drugs and metabolites. Thus, altered hepatic transport protein function (due to disease state, genetic polymorphism or drug-drug interactions) may influence systemic and/or hepatic concentrations, thereby altering therapeutic efficacy and/or toxicity. A multiexperimental approach was employed to address these key issues regarding hepatic transport mechanisms: 1) sex-related differences in the hepatobiliary disposition of phase II conjugates; 2) in vitro tools to assess altered transport function; 3) the role of transport proteins in drug- induced liver injury.

AIM #1. Elucidate the Mechanism of Differences in the Hepatobiliary Disposition of Acetaminophen and Metabolites in Male and Female Mice.

Hypothesis: Sex-dependent hepatic Bcrp expression (male > female) results in sex- dependent hepatobiliary disposition of phase II conjugates. This hypothesis was tested using an in situ mouse liver perfusion approach to compare the disposition of acetaminophen and metabolites in male and female wild-type and Bcrp-deficient mice.

1.a. Characterize the impact of sex-dependent expression of Bcrp on the hepatobiliary disposition of acetaminophen and generated metabolites [glucuronide (AG); sulfate (AS)] in male and female wild-type and Bcrp- deficient mice.

1.b. Use a pharmacokinetic modeling approach to estimate pharmacokinetic parameters and evaluate mechanisms by which the hepatobiliary disposition of Bcrp substrates was altered.

1.c. Compare protein expression levels of the major biliary and basolateral transport proteins among male and female wild-type and Bcrp-deficient mice.

AIM #2. Assess the Utility of an Integrated in vitro Approach [Sandwich- cultured Hepatocytes (SCH) and Modeling/Simulation] in Predicting Alterations in the Hepatobiliary Disposition of Troglitazone (TGZ) and Metabolites.

Hypothesis: Rat and Human SCH can be used to mimic the in vivo disposition of TGZ and generated metabolites in rats and humans. Compartmental pharmacokinetic modeling and Monte Carlo simulation approaches can be applied to data from SCH to examine the consequences of canalicular transport protein modulation resulting from drug interactions or disease states, on hepatic exposure in hypothetical “populations” of hepatocytes.

2.a. Assess the metabolism of TGZ, which is governed primarily by phase II conjugation, and the hepatobiliary disposition of TGZ and generated metabolites in rat and human SCH.

2.b. Use pharmacokinetic modeling and Monte Carlo simulation approaches to predict alterations in hepatic exposure and routes of excretion of TGZ and generated metabolites.

AIM #3. Examine the Potential Role of Hepatic Transport Proteins in Liver Injury Mediated by Trabectedin and Sulindac.

Hypothesis 1: Mrp2 as well as CYP3A1/2 play a role in the previously reported