Hepatic disposition of 5 (and 6)-carboxy-2Ј,7Ј-dichlorofluorescein (CDF) and its diacetate promoiety (CDFDA) was studied in isolated perfused rat livers. Livers from Wistar wild-type and multidrug resistance-associated protein (Mrp)2-deficient (TR Ϫ ) rats were perfused with CDF in the presence or absence of probenecid. Probenecid decreased the recovery of CDF in bile ϳ4-fold in wild-type livers (65 Ϯ 8% versus 15 Ϯ 2% of dose over 2 h). In livers from TR Ϫ rats, CDF was not excreted into bile and probenecid decreased perfusate CDF concentrations in a concentration-dependent manner, in part due to inhibition of Mrp3. Plasma membrane vesicles from rat Mrp2-or Mrp3-transfected Sf9 cells were used to confirm that CDF is a substrate for Mrp2 and Mrp3; probenecid inhibited the transport of CDF by Mrp2 and Mrp3 in a concentration-dependent manner.CDF uptake in collagen sandwich-cultured rat hepatocytes was temperature-dependent and saturable (K m ϭ 22 Ϯ 10 M; V max ϭ 97 Ϯ 9 pmol/min/mg protein). Uptake of CDF in sandwich-cultured rat hepatocytes was impaired significantly by bromosulfophthalein, a substrate for organic anion-transporting polypeptides (Oatps), but was not modulated by specific Oatp2 or organic anion transporter (Oat) substrates. CDFDA uptake was not saturable, temperature-dependent, or impaired by inhibitors. The hydrolysis of CDFDA to CDF is mediated by basic pH and esterases in biological media. CDFDA passively diffuses into hepatocytes where it is hydrolyzed to CDF. In contrast, CDF appears to be taken up by Oatp-mediated transport into rat hepatocytes and effluxed via Mrp2 into bile and via Mrp3 into sinusoidal blood.

The discovery of a pyrrolopyrimidine class of LIM-kinase 2 (LIMK2) inhibitors is reported. These LIMK2 inhibitors show good potency in enzymatic and cellular assays and good selectivity against ROCK. After topical dosing to the eye in a steroid induced mouse model of ocular hypertension, the compounds reduce intraocular pressure to baseline levels. The compounds also increase outflow facility in a pig eye perfusion assay. These results suggest LIMK2 may be an effective target for treating ocular hypertension and associated glaucoma.

The role of Mrp2, Bcrp, and P-glycoprotein in the biliary excretion of acetaminophen sulfate (AS) and glucuronide (AG), 4-methylumbelliferyl sulfate (4MUS) and glucuronide (4MUG), and harmol sulfate (HS) and glucuronide (HG) was studied in Abcc2(Ϫ/Ϫ), Abcg2(Ϫ/Ϫ), and Abcb1a(Ϫ/Ϫ)/Abcb1b(Ϫ/Ϫ) mouse livers perfused with the respective parent compounds using a cassette dosing approach. Biliary clearance of the sulfate conjugates was significantly decreased in Bcrp-deficient mouse livers, resulting in negligible biliary excretion of AS, 4MUS, and HS. It is noteworthy that the most profound decrease in the biliary clearance of the glucuronide conjugates was observed in Bcrp-deficient mouse livers, although the biliary clearance of 4MUG was also ϳ35% lower in Mrp2-deficient mouse livers. As expected, biliary excretion of conjugates was not impaired in P-glycoprotein-deficient livers. An appreciable increase in perfusate recovery due to a shift in the directionality of metabolite excretion, from bile to perfusate, was noted in knockout mice only for conjugates whose biliary clearance constituted an appreciable (Ն37%) fraction of total hepatic excretory clearance (i.e., 4MUS, HG, and HS). Biliary clearance of AG, AS, and 4MUG constituted a small fraction of total hepatic excretory clearance, so an appreciable increase in perfusate recovery of these metabolites was not observed in knockout mice despite markedly decreased biliary excretion. Unlike in rats, where sulfate and glucuronide conjugates were excreted into bile predominantly by Mrp2, mouse Bcrp mediated the biliary excretion of sulfate metabolites and also played a major role in the biliary excretion of the glucuronide metabolites, with some minor contribution from mouse Mrp2.Phase II metabolism, including sulfation and glucuronidation, occurs primarily in the liver. Conjugation of a substrate with a sulfate or glucuronide moiety increases its hydrophilicity to promote excretion from the body. These conjugates are typically too polar to undergo passive diffusion from hepatocytes after their intracellular formation and therefore require carrier-mediated transport for excretion across the hepatic canalicular (apical) membrane into bile and across the basolateral membrane into sinusoidal blood. Most sulfate and glucuronide metabolites are inactive; notable exceptions include morphine-6-glucuronide, minoxidil sulfate, SN-38-glucuronide, and troglitazone sulfate (Zamek-Gliszczynski et al., 2006b). Altered hepatic export of pharmacologically and toxicologically active sulfate and glucuronide metabolites formed in the liver can have profound pharmacodynamic and toxic implications, underscoring the importance of understanding the mechanisms of metabolite excretion (Meisheri et al., 1993;Funk et al., 2001;Horikawa et al., 2002;. Despite the importance of active transport in hepatic excretion of phase II metabolites, mechanisms responsible for excretion of sulfate and glucuronide conjugates have not been elucidated fully.

The structure of LX7101, a dual LIM-kinase and ROCK inhibitor for the treatment of ocular hypertension and associated glaucoma, is disclosed. Previously reported LIM kinase inhibitors suffered from poor aqueous stability due to solvolysis of the central urea. Replacement of the urea with a hindered amide resulted in aqueous stable compounds, and addition of solubilizing groups resulted in a set of compounds with good properties for topical dosing in the eye and good efficacy in a mouse model of ocular hypertension. LX7101 was selected as a clinical candidate from this group based on superior efficacy in lowering intraocular pressure and a good safety profile. LX7101 completed IND enabling studies and was tested in a Phase 1 clinical trial in glaucoma patients, where it showed efficacy in lowering intraocular pressure.

ABSTRACT:This study characterized the hepatobiliary disposition of 5 (and 6)-carboxy-2,7-dichlorofluorescein (CDF), a model Abcc2/Mrp2 (canalicular) and Abcc3/Mrp3 (basolateral) substrate, in perfused livers from male C57BL/6 wild-type, Abcg2؊/؊, and Abcc2؊/؊ mice. After single-pass liver perfusion with 1 M CDF diacetate for 30 min and an additional 30-min perfusion with CDF-free buffer, cumulative biliary excretion of CDF in Abcg2؊/؊ mice was significantly higher than in wild-type mice (65 ؎ 6 and 47 ؎ 15% of dose, respectively, p < 0.05), whereas CDF recovery in bile of Abcc2؊/؊ mice was negligible. Cumulative recovery of CDF in perfusate was significantly higher in Abcc2؊/؊ (90 ؎ 8% of dose) relative to wild-type (

Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1−/− B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1−/− CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.

ABSTRACT:Previous studies implicated P-glycoprotein (P-gp) as the major transport protein responsible for the biliary excretion of fexofenadine (FEX). However, FEX biliary excretion was not impaired in P-gp-or breast cancer resistance protein (Bcrp)-knockout mice or multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The present study tested the hypothesis that species differences exist in the transport protein primarily responsible for FEX biliary excretion between mice and rats. Livers from Mrp2-knockout (Mrp2KO) mice and Mrp2-deficient (TR ؊ ) rats were perfused in a single-pass manner with 0.5 M FEX. N-(4-[2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (10 M) was employed to inhibit P-gp and Bcrp. FEX is a nonsedating H 1 receptor antagonist used clinically for the treatment of seasonal allergies. FEX is metabolically stable; less than 5% of the dose is eliminated by metabolism in humans (Cvetkovic et al., 1999). FEX is eliminated primarily via biliary excretion. In mice, biliary and renal clearances account for 50 to 70% and 10 to 20% of FEX total body clearance, respectively (Tahara et al., 2005).In vitro studies have demonstrated that cellular uptake of FEX is mediated by the 1A2 and 2B1 isoforms of human organic anion transporting polypeptide (OATP1A2 and OATP2B1, respectively); cellular efflux of FEX is mediated by human P-gp (Cvetkovic et al., 1999). FEX concentrations in plasma, brain, kidney, and liver were elevated significantly after oral and intravenous administration to P-gp-knockout mice (Cvetkovic et al., 1999). FEX has been used as a P-gp probe substrate for clinical drug-drug and food-drug interaction studies (Banfield et al., 2002;Shon et al., 2005).In rodents, FEX biliary excretion was not impaired in P-gp-or Bcrp-knockout mice or Mrp2-deficient Eisai hyperbilirubinemic rats (Tahara et al., 2005). These findings led to the hypothesis that one or more unidentified transport protein(s) distinct from P-gp, Mrp2, and Bcrp mediates the biliary excretion of FEX. Recent studies have suggested that species differences exist in transport mechanisms mediating biliary excretion (Zamek-Gliszczynski et al., 2006b). The purpose of this work was to determine whether Mrp2 is responsible for FEX biliary excretion in mice and whether species differences in FEX biliary excretion exist between mice and rats. Furthermore, the involvement of transport mechanisms other than Mrp2, P-gp, and Bcrp in FEX biliary excretion was investigated by using the P-gp and Bcrp inhibitor N- (4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) in Mrp2-knockout mice.

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