Sinusoidal Efflux of Taurocholate Is Enhanced in Mrp2-Deficient Rat Liver

Purpose. It has been shown that plasma concentration and urinary excretion of bile acids is elevated under the cholestatic/hyperbilirubinemic conditions. Previously, it was demonstrated that the plasma concentration of bile acids was elevated in the multidrug resistance-associated protein 2 (Mrp2)-deficient rats. The purpose of the present study was to compare the sinusoidal efflux clearance of taurocholate (TC) between Mrp2-deficient Eisai hyperbilirubinemic rats (EHBR) and normal rats. Method. Hepatic disposition of the [³H]TC was examined in the perfused liver. Apparent efflux clearance (PS_{net, eff}) of [³H]TC from hepatocytes to outflow across the sinusoidal membrane was defined as the amount of [³H]TC excreted into the outflow from the liver divided by hepatic AUC of [³H]TC. Additionally, influx clearance (PS_inf) was also determined by multiple indicator dilution method because PS_{net, eff} is also affected by PS_inf. Results. PS_{net, eff} was significantly higher in EHBR than that in Sprague-Dawley (SD) rats (16.6 ±1.7 vs. 6.1 ± 1.3 L/min/g liver, P <0.01). In contrast, PS_inf was comparable between SD rats and EHBR. Kinetic analysis suggested that the intrinsic clearance for the efflux of [³H]TC across the sinusoidal membrane in EHBR was higher than that in SD rats (10.4 ± 1.0 v.s. 23.3 ± 1.7 L/min/g liver, P <0.01). Conclusions. Enhanced sinusoidal efflux of TC in EHBR may be related to the altered disposition of bile acids in the mutant rats. Because Mrp3 transports TC and its expression is induced on the basolateral membrane of Mrp2-deficient rats, the enhanced sinusoidal efflux of TC in EHBR may be accounted for, at least partially, by the increased expression of Mrp3.     

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats

Abstract

1.?Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions.

2.?Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague–Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs.

3.?An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17β-glucuronide (E₂17βG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides.

4.?In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.     

Pharmacokinetic Study of the Hepatobiliary Transport of Indomethacin

Purpose. The biliary excreted amount of indomethacin and itsglucuronide is related to the intestinal toxicity of this drug. In the presentstudy, we investigated the hepatobiliary transport of indomethacin.Methods. The uptake of indomethacin into primary cultured rathepatocytes and COS-7 cells transfected with cDNA encoding sodiumtauro-cholate co-transporting polypeptide or organic anion transportingpolypeptide 1 was examined. Moreover, we compared the biliaryexcretion of indomethacin and its glucuronide between Sprague-Dawley(SD) rats and Eisai hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective.Results. The uptake of indomethacin into rat hepatocytes was mediatedby Na⁺-dependent and independent active transport systems. Neithertransfectant stimulated the uptake of indomethacin. After intravenousinfusion of indomethacin to SD rats, the biliary excretion ofindomethacin glucuronide exceeded that of indomethacin. The indomethacintransport clearance across the bile canalicular membrane wascomparable between SD rats and EHBR, whereas the corresponding value forindomethacin glucuronide in EHBR was approximately 50% that inSD rats.Conclusions. These results indicate that another transporter(s) isinvolved in the hepatic uptake of indomethacin and the canaliculartransport of indomethacin glucuronide is mediated by cMOAT/MRP2whereas that of indomethacin is not mediated by cMOAT/MRP2.     

Kinetic Analysis of Hepatobiliary Transport for Conjugated Metabolites in the Perfused Liver of Mutant Rats (EHBR) with Hereditary Conjugated Hyperbilirubinemia

Purpose. Previously, we found that the biliary excretion of the 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040) glucuronide is severely impaired in Eisai hyperbilirubinemic rats (EHBR), while that of sulfate remains normal (Takenaka et al., J. Pharmacol. Exp. Then, 274: 1362–1369, 1995). The purpose of the present study is to clarify the mechanisms for impairment of the biliary excretion of E3040 glucuronide in EHBR. Methods. We kinetically analyzed the disposition of the conjugates in the perfused liver at steady state. The uptake of the conjugates into the isolated canalicular membrane vesicles (CMVs) was also examined. Results. At steady state, the bile/liver unbound concentration ratios of the conjugates were 40-400 in both rat strains, indicating a highly concentrated process. The biliary excretion clearance (CL_u,bile) of the glucuronide, defined for the unbound concentration in the liver, was decreased in EHBR to 1/30 of that in normal rats, whereas the CL_u,bile of the sulfate was comparable between the two rat strains. In vitro, the transport of E3040 glucuronide into CMV prepared from SD rats exhibited the ATP dependency, whereas minimal effect of ATP was observed on the uptake of the glucuronide into CMV from EHBR. In contrast, the uptake of E3040 sulfate was comparable between SD rats and EHBR. Furthermore, ATP did not stimulate the uptake of sulfate into the CMVs. Conclusions. It was suggested (1) that the excretion of E3040 glucuronide across the bile canalicular membrane is mediated by the primary active transporter which is defective in EHBR and (2) that the bile canalicular transport system for E3040 sulfate is different from that for the glucuronide in that the former remains normal in EHBR.     

Comparative Inhibitory Effects of Different Compounds on Rat Oatp1 (Slc21a1)- and Oatp2 (Slc21a5)-Mediated Transport

Purpose. The purpose of the present study is to examine the selectivity of various inhibitors towards the rat organic anion transporting polypeptides 1 (Oatp1: gene symbol Slc21a1) and 2 (Oatp2: Slc21a5). Methods. The inhibitory effects of 20 compounds on the Oatp1-mediated transport of estradiol 17-D-glucuronide and on the Oatp2-mediated transport of digoxin were examined in cDNA-transfected LLC-PK₁ cells. Results. Among the compounds examined in this study, nonsteroidal anti-inflammatory drugs, deoxycorticosterone, and quinidine preferentially inhibited Oatp1, whereas digoxin, quinine, and rifampicin preferentially inhibited Oatp2 at low concentrations. On the other hand, propionic acid, -ketoglutarate and p-aminohippurate showed no inhibitory effects on either transporter up to a concentration of 1000 M. The K_i values of ibuprofen and quinidine were estimated to be 19 and 13 times lower for Oatp1 compared with Oatp2, whereas the values for rifampicin, quinine, and digoxin were 13, 20, and 100< times lower for Oatp2 compared with Oatp1. Conclusions. At low concentrations, some of the tested inhibitors exert selective inhibition of either Oatp1- or Oatp2-mediated substrate transport. These selective inhibitors may be used at appropriate concentrations to estimate the maximum contribution of Oatp1 or Oatp2 to the total substrate uptake into rat hepatocytes.     

The 5-HT1A receptor selective ligands, (R)-8-OH-DPAT and (S)-UH-301, differentially affect the activity of midbrain dopamine neurons

Summary The effects of the selective 5-HT_1A receptor agonist (R)-8-hydroxy-2(di-n-propylamino)tetralin [(R)-8-OH-DPAT] and the novel 5-HT_1A antagonist (S)-5-fluoro-8-hydroxy-2-(dipropylamino)-tetralin [(S)-UH-301] were studied with regard to the firing pattern of single mesencephalic dopamine (DA) neurons with extracellular recording techniques in chloral hydrate anesthetized male rats. Neuronal activity was studied with respect to firing rate, burst firing and regularity of firing. In the ventral tegmental area (VTA) low doses of (R)-8-OH-DPAT (2–32 g/kg i.v.) caused an increase in all three parameters. The effect on firing rate of DA neurons was more pronounced in the parabrachial pigmentosus nucleus than in the paranigral nucleus, the two major subdivisions of VTA. In the substantia nigra zona compacta (SN-ZC), (R)-8-OH-DPAT (2–256 g/kg i.v.) had no effect on firing rate and regularity of firing and only slightly increased burst firing. High doses of (R)-8-OH-DPAT (512–1024 g/kg i.v.) decreased the activity of DA cells in both areas, an effect that was prevented by pretreatment with the selective DA D₂ receptor antagonist raclopride. (S)-UH-301 (100–800 g/kg i.v.) decreased both firing rate and burst firing without affecting regularity of DA neurons in the VTA. In the SN-ZC, (S)-UH-301 decreased the firing rate but failed to affect burst firing and regularity of firing. These effects of (S)-UH-301 were blocked by raclopride pretreatment. Local application by pneumatic ejection of 8-OH-DPAT excited the DA cells in both the VTA and the SN-ZC, whereas (S)-UH-301 inhibited these cells when given locally. These results show that 5-HT_1A receptor related compounds differentially affect the electrophysiological activity of central DA neurons. The DA receptor agonistic properties of these compound appear to contribute to the inhibitory effects of high doses of (R)-8-OH-DPAT and (S)-UH-301 on DA neuronal activity. Given the potential use of 5-HT_1A receptor selective compounds in the treatment of anxiety and depression their effects on central DA systems involved in mood regulation and reward related processes are of considerable importance.Correspondence to T. H. Svensson at the above address     

Partial Maintenance of Taurocholate Uptake by Adult Rat Hepatocytes Cultured in a Collagen Sandwich Configuration

Purpose. This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. Methods. Hepatocytes isolated from male Wistar rats (230–280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein, Na⁺/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. Results. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na⁺-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 M) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent K_m values were comparable for day-0 and day-5 hepatocytes (32–41 M). In contrast, the V_max values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. Conclusions. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.     

Metabolic Studies with Trans-5-Amino-3-[2-(5-Nitro-2-Furyl)Vinyl]-1,2,4-[5-14C]Oxadiazole (SQ 18,506): 1. Reductive Cleavage of the 1, 2, 4-oxadiazole ring

Abstract

1. Reductive cleavage of the oxadiazole ring of trans-5-amino-3-[2-(5-nitro-2-furyl)vinyl]-1,2,4-[5-¹⁴C]oxadiazole (SQ 18,506-¹⁴C) has been demonstrated both enzymically and chemically.

2. Oral administration of SQ 18,506-¹⁴C to humans and rat resulted in the formation of respiratory ¹⁴CO₂ and the excretion of ¹⁴C-urea in the urine.

3. Formation of ¹⁴CO₂ was demonstrated in vitro by incubation of SQ 18,506-¹⁴C with homogenate of human or mouse liver or with milk xanthine oxidase.

4. ¹⁴C-urea was isolated after reduction of SQ 18,506-¹⁴C by ferrous hydroxide.     

Transport,Uptake, and Metabolism of the Bis(pivaloyloxymethyl)-Ester Prodrug of 9-(2- Phosphonylmethoxyethyl)Adenine in an In Vitro Cell Culture System of the Intestinal Mucosa (Caco-2)

Purpose. To evaluate intestinal transport, uptake and metabolism characteristics of the bis(pivaloyloxymethyl)-ester [bis(POM)-ester] of the antiviral agent 9-(2-phosphonylmethoxyethyl)adenine [PMEA]. Methods. Intestinal transport, uptake and metabolism of bis(POM)-PMEA were studied using an in vitro cell culture system of the intestinal mucosa (Caco-2 monolayers). Concentrations of bis(POM)-PMEA and its metabolites mono(POM)-PMEA and PMEA were determined using a reversed-phase HPLC method. Enzymatic stability of bis(POM)-PMEA was evaluated by incubation with purified liver carboxylesterase, homogenates of Caco-2 cells and scraped pig small intestinal mucosa. Results. The use of bis(POM)-PMEA as a prodrug of PMEA resulted in a significant increase in transport of total PMEA [bis(POM)-PMEA, mono(POM)-PMEA and PMEA] across Caco-2 monolayers. While transepithelial transport of PMEA (500 M) was lower than 0.1% during a 3 hr incubation period, transport of total PMEA after addition of bis(POM)-PMEA (100 M) amounted to 8.8% over the same incubation period. Only 23% of the amount transported appeared as intact bis-ester at the basolateral side, while 33% of this amount was free PMEA and 44% was mono(POM)-PMEA, suggesting susceptibility of the prodrug to chemical and enzymatic degradation. Uptake studies revealed that only negligible amounts of bis(POM)-PMEA (< 0.2%) were present inside the cells. Very high intracellular concentrations of PMEA were found 1.2 mM, after a 3 hr incubation with 50 M bis(POM)-PMEA), which suggests that PMEA was trapped inside the cells probably due to its negative charge. This explains that efflux of PMEA was relatively slow (25% of the intracellular amount in 3 hr). Enzymatic degradation of the prodrug by carboxylesterase was confirmed by incubation of bis(POM)-PMEA with purified enzyme (K_m = 87 M and V_max = 9.5 M/min). Incubation of bis(POM)-PMEA (10 M) with cell homogenate of Caco-2 monolayers and pig small intestinal mucosa produced similar degradation profiles. Conclusions. The use of the bis(POM)-prodrug significantly enhances the intestinal permeability of PMEA. Intracellular trapping of PMEA in the intestinal mucosa may result in slow release of PMEA to the circulation after oral administration of bis(POM)-PMEA.     

Synthesis and anti-HIV evaluation of the novel 2-(m-chlorobenzyl)-4-substituted-7-methyl-1, 1, 3-trioxo-pyrazolo[4, 5-e] [1, 2, 4]thiadiazines

A novel series of 2-(m-Chlorobenzyl)-4-substituted-1, 1, 3-trioxo-2H,4H-pyrazolo[4, 5-e][1, 2, 4] thiadiazines (7a-k) were synthesized, and evaluated for their anti-HIV replication in MT-4 cell cultures. Compound (7a) showed activity against HIV-1-induced cytopathicity, with an EC₅₀ value of 45.6 μM, but none of the compounds exhibited inhibitory activity against HIV-2.     

Pravastatin,an HMG-CoA Reductase Inhibitor,Is Transported by Rat Organic Anion Transporting Polypeptide,oatp2

Purpose. We previously demonstrated the HMG-CoA reductase inhibitor, pravastatin, is actively taken up into isolated rat hepatocytes through multispecific organic anion transporters. The present study examined whether a newly cloned organic anion transporting polypeptide (oatp2) transports pravastatin. Methods. We investigated functional expression of oatp2 in Xenopus laevis oocytes, to examine [¹⁴C] pravastatin uptake. Results. [¹⁴C] Pravastatin (30 M) uptake into oatp2 cRNA-injected oocytes was 40 times higher than that of water-injected control oocytes. The oatp2-mediated pravastatin uptake was Na⁺-independent and saturable. The Michaelis-Menten constant was 37.5 ± 9.9 M, a level comparable to that obtained in isolated rat hepatocytes in our previous study. As is the case with rat hepatocytes, the uptake of pravastatin (30 M) was inhibited by 300 M concentrations of taurocholate, cholate, bromosulfophthalein, estradiol-17-glucuronide, and simvastatin acid, but not by para-aminohippurate. On the other hand, [¹⁴C] simvastatin acid (30 M) uptake of oatp2 cRNA-injected oocytes was not significantly different from that of water-injected oocytes. Conclusions. The cloned oatp2 was identified as the transporter responsible for the active hepatocellular pravastatin uptake.     

Characterization of Hepatobiliary Transport Systems of a Novel α4β1/α4β7 Dual Antagonist, TR-14035

Purpose Our previous pharmacokinetic studies have demonstrated that TR-14035, a novel dual antagonist for α4β1/α4β7 integrin, selectively and strongly accumulated in the liver and was mainly excreted in bile as an unchanged drug. In the present study, we investigated the hepatobiliary transport system in detail.Materials and Methods Uptake by hepatocytes and organic anion transporting polypeptide (OATP)-expressing Xenopus laevis oocytes or Flp-In-293 cells was performed in vitro. Biliary excretion was investigated in mdr1a/b-knockout mice, Bcrp-knockout mice and Mrp2-defective Eisai hyperbilirubinemic rats (EHBRs).Results TR-14035 was taken up by rat and human hepatocytes by an apparently single saturable mechanism with K _m of 6.7 and 2.1 μM, respectively, and taurocholate and digoxin reduced this uptake. OATP1B1/OATP-C and OATP1B3/OATP8 expressed in oocytes mediated the TR-14035 uptake with K _m of 7.5 and 5.3 μM, respectively. OATP1B1*15, a genetic variant of OATP1B1, exhibited a decreased transport of TR-14035 compared with OATP1B1*1a. Biliary excretion and total body clearance of unchanged TR-14035 in EHBRs were significantly lower than those in normal rats, while there was no difference in the clearances between wild and mdr1a/b- or Bcrp-knockout mice.Conclusion These results indicate that OATP1B1 and OATP1B3 are at least partly responsible for the accumulation of TR-14035 into hepatocytes, and Mrp2 principally mediates the biliary excretion of TR-14035. Furthermore, genetic polymorphisms of OATP1B1 may cause an interindividual variability in the pharmacokinetics of TR-14035.     

Enantioselective synthesis of (R)- and (S)-2-methyl-[3,3,2-2H3] alanines

The synthesis of optically pure (R)- and (S)-2-methyl-[3,3,3-²H₃] alanines of biological interest is described. The stereochemistry of the reaction of the lithio derivative of (R)-(©)-2,5-dimethoxy-3-benzyl-3-methyl-3, 6-dihydropyrazine with alkyl and deuterated alkyl iodides is discussed. The configuration of the newly formed center of chirality in (R)- and (S)-2-methyl-[3,3,3-²H₃] alanines is derived from ¹ H NMR.     

Synthesis and biological activity of substituted 7-aza-8-aza(oxa)-bicyclo[4.3.0]-6,9-nonadienes

Hydroxylamine- and hydrazine-induced heterocyclization of readily available 5-hydroxy-2.4-diacetyl-5-methyl-3R-cyclohexanones (R = methyl, phenyl, m-nitrophenyl, -furyl) yielded 6-acetyl-5-hydroxy-5.9-dimethyl-7R-1-aza(oxa)-2-azabicyclo[4.3.0]-2,8-nonadienes (R = methyl, phenyl,m-nitrophenyl. -furyl). The biological activity of the latter was tested using theEscherichia coli phage T₄, and was also tested on a stored lyophilized culture ofYersinia pestis EU.     

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain

Binding characteristics of a novel radioiodinated ligand, [¹²⁵I]R(+)trans-7-hydroxy-2-(N-n-propylN-3-iodo-2-propenyl) aminotetralin (¹²⁵I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [¹²⁵I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (K_d = 0.42 nM). A majority (70%) of the sites labeled by [¹²⁵I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to 5-HT_1A receptors, with a K_d value of 1.4 nM and a B_max value of 210 fmol/mg protein, was observed. Binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D 2 and 5-HT_1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [¹²⁵I]R(+)trans-7-OH-PIPAT. [¹²⁵I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D 3 receptors.Abbreviations 7-OH-DPAT 7-hydroxy-2-N,N-(di-n-propyl)aminotetralin - trans-7-OH-PIPAT traps-7-hydroxy-2-(N-n-propyl-N-3-iodo-2propenyl)aminotetralin - 8-OH-DPAT 8-hydroxy-2-N,N-(di-n-propyl)aminotetralin - 8-OH-PIPAT trans-8-hydroxy-2-(N-n-propyl-N-3-iodo-2-propenyl)aminotetralin - NCQ298 S(–)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy-5,6-dimethoxybenzamide - DTG 1,3-diortho-tolyl-guanidine - Sf9 cells Spodoptera frugiperda insect cells - CHO cells Chinese hamster ovary cells - GTP guanosine 5-triphosphate - WB4101 2-(2,6-dimeethoxyphenoxyethyl)aminomethyl-1,4benzodioxane - R(+)3PPP R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine - (+)MK-801 (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine     

Lipid Association Increases the Potency Against Primary Medulloblastoma Cells and Systemic Exposure of l-(2-Chloroethyl)-3-Cyclohexyl-1-Nitrosourea (CCNU) in Rats

Purpose. To reduce the systemic toxicity and prolong the systemic presence of l-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU), a lipid-based drug carrier was designed and characterized. Methods. The degree of CCNU association with lipid vesicles composed of 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC) and l,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) (1:1, m/m) was characterized and the drug decomposition rates of lipid-drug complexes were monitored. Effects of lipid association on drug potency against medulloblastoma cells and total systemic drug exposure in rats were determined. Results. At a CCNU:lipid molar ratio greater than 1:5, more than 90% of the drug was associated with the lipid vesicles. In aqueous suspensions, lipid association significantly reduced the first-order drug decomposition rate. In addition, lipid-associated CCNU exhibited a 4-fold increase in drug sensitivity with medulloblastoma cells. IC₅₀ values for CCNU admixed and encapsulated with lipid vesicles were 18 ± 4.9 and 14.0 ± 2.2 M, respectively, compared to 83 ± 11.0 M for free CCNU. When administered to rats, lipid-associated CCNU increased the AUC (area under the concentration-time curve) of CCNU by approximately 2-fold (20.46 ± 2.15 compared to 39.59 ±1.87 gmin/ml), and the terminal half-life (t_1/2) by almost 9-fold (17 ± 9 compared to 147 ± 48 min) over free CCNU. Despite the increase in total systemic drug exposure, rats treated with lipid-associated CCNU exhibited a significantly lower frequency of acute neurotoxicity. Conclusions. These data indicate that CCNU associated with lipid vesicles may increase drug stability, potency, and systemic exposure in rats.     

Impact of Mrp2 on the Biliary Excretion and Intestinal Absorption of Furosemide,Probenecid, and Methotrexate Using Eisai Hyperbilirubinemic Rats

Purpose. This study assesses the impact of rat multidrug resistance-associated protein 2 (Mrp2) on the biliary excretion and oral absorption of furosemide, probenecid, and methotrexate using Eisai hyperbilirubinemic rats (EHBR). Methods. To assess Mrp2-mediated biliary excretion, rats received a 2-h intravenous infusion of furosemide, probenecid, or methotrexate. Blood and bile samples were collected at specified intervals. To assess Mrp2's impact on oral absorption, rats received furosemide, probenecid, or methotrexate orally at 5 mg/kg. Jugular and portal blood samples were obtained at timed intervals. All samples were analyzed by LC-MS/MS. Pharmacokinetic parameters were estimated using WinNonlin and standard pharmacokinetic equations. Results. Thirty seven- and 39-fold reductions in biliary clearance were observed in EHBR as compared to control rats for probenecid and methotrexate, respectively. Biliary clearance was comparable between EHBR and control rats for furosemide. In all cases, no significant difference in absorption was observed between EHBR and control rats. Conclusions. This study provides the first evidence that Mrp2 mediates the biliary excretion of probenecid but not furosemide. Additionally, Mrp2 apparently has a less profound impact on intestinal absorption than biliary excretion of its substrates. Furthermore, alteration in systemic clearance in EHBR indicates that a potential compensatory mechanism may occur in EHBR.     

Preclinical assessment of the absorption,distribution, metabolism and excretion of GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide), an orally bioavailable systemic Hedgehog signalling pathway inhibitor

1. GDC-0449 (2-chloro-N-(4-chloro-3-(pyridin-2-yl)phenyl)-4-(methylsulfonyl)benzamide) is a potent, selective Hedgehog (Hh) signalling pathway inhibitor being developed for the treatment of various cancers.

2. The in vivo clearance of GDC-0449 was estimated to be 23.0, 4.65, 0.338, and 19.3?ml min^?1 kg^?1 in mouse, rat, dog and monkeys, respectively. The volume of distribution ranged from 0.490 in rats to 1.68 l kg^?1 in mice. Oral bioavailability ranged from 13% in monkeys to 53% in dogs. Predicted human clearance using allometry was 0.096–0.649?ml min^?1 kg^?1 and the predicted volume of distribution was 0.766 l kg^?1.

3. Protein binding was extensive with an unbound fraction less than or equal to 6%, and the blood-to-plasma partition ratio ranged from 0.6 to 0.8 in all species tested. GDC-0449 was metabolically stable in mouse, rat, dog and human hepatocytes and had a more rapid turnover in monkey hepatocytes.

4. Proposed metabolites from exploratory metabolite identification in vitro (rat, dog and human liver microsomes) and in vivo (dog and rat urine) include three primary oxidative metabolites (M1–M3) and three sequential glucuronides (M4–M6). Oxidative metabolites identified in microsomes M1 and M3 were formed primarily by P4503A4/5 (M1) and P4502C9 (M3).

5. GDC-0449 was not a potent inhibitor of P4501A2, P4502B6, P4502D6, and P4503A4/5 with IC₅₀ estimates greater than 20?μM. K_i’s estimated for P4502C8, P4502C9 and P4502C19 and were 6.0, 5.4 and 24?μM, respectively. An evaluation with Simcyp® suggests that GDC-0449 has a low potential of inhibiting P4502C8 and P4502C9. Furthermore, GDC-0449 (15?μM) was not a potent P-glycoprotein/ABCB1 inhibitor in MDR1-MDCK cells.

6. Overall, GDC-0449 has an attractive preclinical profile and is currently in Phase II clinical trials.

     

Derivatives of 4-(2-N,N-Di-n-propylaminoethyl)-5-hydroxyindole: Synthesis and Pharmacological Effects

5-Methoxy-l-methyl-4-(2-N,N-di-n-propylaminoethyl)indole (12) was synthesized from 5-hydroxyindole by a multistep synthesis. This target compound was designed as a bioisostere of p-dimethoxy catechol congeners of dopaminergic agonists derived from a variety of ring systems, in some of which p-dimethoxy-substituted systems are potent, active dopaminergic agonists. To complete the indole series, all possible combinations of N- and O-demethylated derivatives of 12 were prepared and were also evaluated pharmacologically. All members of this indole-derived series showed a low order of cardiovascular activity, which appeared to be independent of dopamine receptors. The lack of dopaminergic activity of 12 is cited as yet another example of the unpredictable effect of replacement of the catechol moiety of a dopaminergic agonist with a p-dimethoxy moiety.To whom correspondence should be addressed.     

Synthesis of 7-[p-(methylthio)benzoyl]-5-benzofuranacetic acid

A new method was described for the preparation of 7-[p-(methylthio)benzoyl]-5-benzofuranacetic acid6, which is an analgesic agent. Methyl 5-(2,3-di-hydrobenzofuran)acetate3 was obtained by Friedel-Crafts reaction of 2,3-dihydrobenzofuran with methyl α-chloro-α-(methylthio)actate1 and desulfurization of2. Tifurac6 was synthesized from acylation of 3 with p-(methylthio)benzoyl chloride followed by bromination of4, dehydrohalogenation, and hydrolysis of5.