Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line

Purpose

The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin.

Methods

Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis.

Results

Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a K_m of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (K_m?=?0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA.

Conclusions

Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

     

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.     

Interaction of Ibuprofen and Other Structurally Related NSAIDs with the Sodium-Coupled Monocarboxylate Transporter SMCT1 (SLC5A8)

Purpose Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na⁺-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). Methods We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [¹⁴C]-nicotinate and [³H]-ibuprofen for direct measurements of uptake in oocytes. Results In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na⁺ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 ± 16 μM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na⁺ dependent, and competitive. However, there was no uptake of [³H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [¹⁴C]-nicotinate was demonstrable under identical conditions. Conclusions Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.     

Transport of Nicotinate and Structurally Related Compounds by Human SMCT1 (SLC5A8) and Its Relevance to Drug Transport in the Mammalian Intestinal Tract

Purpose To examine the involvement of human SMCT1, a Na⁺-coupled transporter for short-chain fatty acids, in the transport of nicotinate/structural analogs and monocarboxylate drugs, and to analyze its expression in mouse intestinal tract. Materials and Methods We expressed human SMCT1 in X. laevis oocytes and monitored its function by [¹⁴C]nicotinate uptake and substrate-induced inward currents. SMCT1 expression in mouse intestinal tract was examined by immunofluorescence. Results [¹⁴C]Nicotinate uptake was several-fold higher in SMCT1-expressing oocytes than in water-injected oocytes. The uptake was inhibited by short-chain/medium-chain fatty acids and various structural analogs of nicotinate. Exposure of SMCT1-expressing oocytes to nicotinate induced Na⁺-dependent inward currents. Measurements of nicotinate flux and associated charge transfer into oocytes suggest a Na⁺:nicotinate stoichiometry of 2:1. Monocarboxylate drugs benzoate, salicylate, and 5-aminosalicylate are also transported by human SMCT1. The transporter is expressed in the small intestine as well as colon, and the expression is restricted to the lumen-facing apical membrane of intestinal and colonic epithelial cells. Conclusions Human SMCT1 transports not only nicotinate and its structural analogs but also various monocarboxylate drugs. The transporter is expressed on the luminal membrane of the epithelial cells lining the intestinal tract. SMCT1 may participate in the intestinal absorption of monocarboxylate drugs.     

Transport of 2,4-dichloro phenoxyacetic acid by human Na+-coupled monocarboxylate transporter 1 (hSMCT1, SLC5A8)

Using X. laevis oocyte expression system, we investigated whether human Na⁺-coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na⁺-dependent and electrogenic. The Na⁺-dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na⁺-dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na⁺-dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity.     

Amino Acids,XII: (±)Pipecolic Acid Derivatives - Part 2: An Expedient Synthetic Entry to Substituted Pipecolic Acids

The Diels-Alder product 1 is transformed by Noyori reaction and catalytic hydrogenation to 4 . Hydrolysis with concomitant decarboxylation of 4 furnishes the trans configuration amino acid 5 . Functional group transformation (reduction, lactonization) of 5 provides 7 , ring opening affords the pipecolic acid derivative 9 . On the other hand 1 is hydrolyzed to 10 and 4-oxo-pipecolic acid 11 . Reduction of 10 and subsequent hydrolysis with decarboxylation of 12 affords the amino acid 13a /13b in a cis/trans ratio of 1:1. Contrary to this result, reduction of 11 provides 13a/13b in a ratio of ≧ 95/5.     

Putative Irreversible Inhibitors of the Human Sodium-Dependent Bile Acid Transporter (hASBT; SLC10A2) Support the Role of Transmembrane Domain 7 in Substrate Binding/Translocation

Purpose

To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes.

Methods

Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting model). The ability of electrophilic derivatives to interact with hASBT was evaluated by 2-aminoethyl-methanethiosulfonate (MTSEA)-biotin labeling of thiol groups in TM7 cysteine mutants.

Results

Unlike native BAs, the electrophilic CDCA derivatives specifically inactivated hASBT, but not hOCTN2, and inhibited hASBT in a time- and concentration-dependent fashion. Preincubation of hASBT Cys-mutants in the exofacial half of TM7 with reactive electrophilic probes blocked transporter biotinylation by MTSEA-biotin, similar to 2-(trimethylammonium)ethyl-methanethiosulfonate (MTSET) blocking. This blocking pattern differed from that produced by native BAs, which exposed exofacial TM7 residues, thereby increasing staining.

Conclusion

Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the role of TM7 in binding/translocation of bile acids.     

Human organic anion transporters 1 (hOAT1/SLC22A6) and 3 (hOAT3/SLC22A8) transport edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one) and its sulfate conjugate.

3-Methyl-1-phenyl-2-pyrazolin-5-one (MCI-186; edaravone), a novel free radical scavenger, is used for the treatment of acute cerebral infarction. After marketing, a few cases of acute renal failure were reported in patients following treatment with this drug. Because edaravone is mainly excreted into the urine following conjugation to glucuronide or sulfate, the renal excretion mechanisms of edaravone should help provide important information when considering the clinical cases. We examined the transport of edaravone and its sulfate and glucuronide conjugates via human organic anion transporter 1 (hOAT1) and 3 (hOAT3), expressed on the basolateral membranes of proximal tubules. The hOAT1- and hOAT3-transfected human embryonic kidney (HEK)-293 cells exhibited a markedly higher uptake of edaravone sulfate and a slightly higher uptake of edaravone than vector-transfected cells. The K(m) values of edaravone sulfate uptake by hOAT1 and hOAT3 were 11 and 15 microM, respectively. Estimation of the relative contribution of hOAT1 and hOAT3 using reference compounds suggested that hOAT1 and hOAT3 might contribute to the renal uptake of edaravone sulfate to the same extent. However, edaravone and its sulfate showed no cytotoxicity toward both hOAT1-HEK and control cells, suggesting that higher uptake in hOAT1-HEK did not associate with cytotoxicity of these compounds. In conclusion, our results suggest that both hOAT1 and hOAT3 are responsible for the basolateral uptake of edaravone sulfate in the kidney.     

Amino Acids,XIV; (±)-Pipecolic Acid Derivatives,IV: An Efficient Synthetic Method for the Preparation of (±)-Baikiain and its Derivatives

[4+2] Cycloaddition of the highly electrophilic imine 2 to 1,3-butadiene ( 1 ) furnished the Diels-Alder product 3 . Hydrolysis of the geminal diester provided (±)-Baikiain hydrochloride ( 4 ). Cis-Hydroxylation of the double bond of 3 afforded 8 . The trans product 9 was prepared via ring opening of the epoxide 5 . The regioselective hydroboration/oxidation of 3 provided 7 which was oxidized to 11 . Halolactonization of the N-tosyl derivative 12 of Baikiain furnished after functional group transformation the 2-hydroxymethyl-piperidine-4-ol 15 .     

Involvement of carnitine/organic cation transporter OCTN2 (SLC22A5) in distribution of its substrate carnitine to the heart

This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na(-)-independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.     

UPLC-MS/MS检测大鼠血浆中沃诺拉赞、代谢产物沃诺拉赞羧酸(M1)及其药动学研究

目的 建立一种同时快速检测大鼠血浆中沃诺拉赞及其代谢产物沃诺拉赞羧酸(M1)的超高效液相色谱串联质谱方法(UPLC-MS/MS),并应用该方法开展其在大鼠体内的药动学研究。方法 使用ACQUITY UPLC^® BEH C₁₈柱(100 mm×2.1 mm, 1.7 μm)对沃诺拉赞和M1进行分离,柱温为40 ℃;流动相为乙腈-水(含0.1%甲酸),梯度洗脱,流速0.4 mL·min^–1;采用ESI⁺电喷雾离子源结合多反应监测模式进行检测,沃诺拉赞的定量离子对为m/z 346.04→314.97,代谢产物M1的离子对为m/z 347.08→205.06;大鼠血浆加入内标后经乙腈沉淀去除蛋白,离心后取2 μL进样。所有数据应用DAS 3.2.7软件进行分析得到药动学参数。结果 沃诺拉赞和M1的保留时间分别为1.07 min和1.25 min;标准曲线显示沃诺拉赞和M1分别在5~1 000 ng·mL^–1和10~2 000 ng·mL^–1内呈良好线性关系;沃诺拉赞和M1的精密度和准确度为–4.41%~11.68%,提取回收率为78.85%~86.05%,基质效应为98.54%~104.08%;沃诺拉赞和M1的稳定性结果RSD均< 15.0%。大鼠灌胃10 mg·kg^–1沃诺拉赞后,体内药物和代谢产物M1的曲线下面积AUC_(0~t)分别为1 972.51,13 232.42 μg·L^–1·h,半衰期t_1/2分别为2.97,2.13 h,血浆清除率CLz分别为5.13,0.76 L·h^–1·kg^–1。结论 该方法分析时间短、操作简便,方法学均符合生物样品分析相关要求,可以适用于大鼠体内沃诺拉赞及其代谢产物M1浓度检测和药动学研究。     

Hepatobiliary Transport of a Nonpeptidic Endothelin Antagonist, (+)-(5S,6R, 7R)-2-Butyl-7-[2((2S)-2-Carboxypropyl)-4-Methoxyphenyl]-5-(3,4-Methylenedioxyphenyl) Cyclopentenol[1,2-b]Pyridine-6-Carboxylic Acid: Uptake by Isolated Rat Hepatocytes and Canalicular Membrane Vesicles

Purpose. Hepatobiliary excretions of drugs from the blood to the bile include two essential transmembrane processes: uptake into hepatocytes and secretion from hepatocytes. The purpose of this study was to clarify the transport mechanisms underlying these processes for a new non-peptide endothelin antagonist, (+)-(5S,6R,7R)-2-butyl-7-[2((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxy- phenyl)cyclopentenol[1,2-b]pyridine-6-carboxylic acid (J-104132). Methods. Biliary excretion of J-104132 was assessed in rats after intravenous injection. To evaluate the hepatic uptake process, J-104132 was incubated with freshly isolated rat hepatocytes and the uptake of J-104132 was calculated. To evaluate the biliary secretion process, the uptake of J-104132 into rat canalicular membrane vesicles that were isolated from normal Sprague-Dawley rats or Eisai hyperbilirubinemic rats was measured. Results. After intravenous injection, J-104132 was recovered from the bile quantitatively (99.7 ± 1.3%) as its intact form. J-104132 was taken up by isolated rat hepatocytes in a time- and temperature-dependent manner. The uptake was saturable with K _m and V _max of 5.7 M and 564 pmol/min/10⁶ cells, respectively. The uptake was Na⁺ independent and was reduced in the presence of ATP depleters (rotenone and carbonyl cyanide-p-(trifluoromethoxy)-phenylhydra- zone), organic anions (dibromosulfophthalein, indocyanine green, BQ-123, and pravastatin), and bile acids (taurecholate and cholate). In Sprague-Dawley rats, J-104132 was taken up by canalicular membrane vesicle ATP-dependently with K_m and V_max values of 6.1 M and 552 pmol/min/mg protein, respectively. However, ATP-dependent uptake disappeared in Eisai hyperbilirubinemic rats. Conclusions. These data suggest that energy-dependent and carrier-mediated transport systems play important roles in hepatobiliary excretion of J-104132 (both uptake and secretion processes), which is the main excretion route in rats. As for the secretion process of J-104132, an involvement of mrp2 was demonstrated.     

N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) interaction with LYS 3.28(192) is crucial for its inverse agonism at the cannabinoid CB1 receptor

In superior cervical ganglion neurons, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A) competitively antagonizes the Ca(2+) current effect of the cannabinoid (CB) agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN55212-2), and behaves as an inverse agonist by producing opposite current effects when applied alone. In contrast, in neurons expressing CB1 with a K-->A mutation at residue 3.28(192) (i.e., K3.28A), SR141716A competitively antagonizes the effects of WIN55212-2, but behaves as a neutral antagonist by producing no current effects itself. Receptor modeling studies suggested that in the CB1 inactive (R) state, SR1417A16A stabilizes transmembrane helix 6 in its inactive conformation via aromatic stacking with F3.36/W6.48. In this binding site, SR141716A would exhibit higher affinity for CB1 R due to a hydrogen bond between the SR141716A C3 substituent and K3.28(192), a residue available to SR141716A only in R. To test this hypothesis, a "mutant thermodynamic cycle" was constructed that combined the evaluation of SR141716A affinity at WT CB1 and K3.28A with an evaluation of the wild-type CB1 and K3.28A affinities of an SR141716A analog, 5-(4-chlorophenyl)-3-[(E)-2-cyclohexylethenyl]-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole (VCHSR), that lacks hydrogen bonding potential at C3. Binding affinities suggested that K3.28 is involved in a strong interaction with SR141716A in WT CB1, but does not interact with VCHSR. Thermodynamic cycle calculations indicated that a direct interaction occurs between the C3 substituent of SR141716A and K3.28 in WT CB1. Consistent with these results, VCHSR acted as a neutral antagonist at WT CB1. These results support the hypothesis that hydrogen bonding of the SR141716A C3 substituent with K3.28 is responsible for its higher affinity for the inactive R state, leading to its inverse agonism.     

Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy

Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b₅ alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine–DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b₅ enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine–DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine–DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b₅ might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b₅ in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b₅ and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.     

Intrinsic activity of enantiomers of 8-hydroxy-2-(di-n-propylamino)tetralin and its analogs at 5-hydroxytryptamine1A receptors that are negatively coupled to adenylate cyclase

Although many different types of compounds have been tested for 5-hydroxytryptamine1A (5-HT1A) binding affinity, much remains to be learned about the structural requirements associated with 5-HT1A agonism, partial agonism, and antagonism. The present study uses the forskolin-stimulated adenylate cyclase (FSC) assay as a functional screen in rat hippocampal membranes to examine structure-activity relationships for a series of enantiomers of novel analogs of the prototypic 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The findings illustrate that there can be large enantiomeric differences in intrinsic activity at the 5-HT1A receptor, independent of enantiomeric effects on binding affinity. Generally, for each enantiomeric pair exhibiting stereoselective 5-HT1A binding, the enantiomer with the higher affinity also displayed the greater amount of 5-HT1A intrinsic activity in the FSC assay. Interestingly, the enantiomers of 8-OH-DPAT itself displayed stereoselective differences in intrinsic activity but not 5-HT1A affinity. Several of the compounds, namely (S)-UH-301, (2R,3R)-CM-12, and (1S,2R)-LEA-146, may have potential as prototypes for selective 5-HT1A antagonists, and (S)-UH-301 itself may be useful as a selective 5-HT1A antagonist. The FSC data presented here are in good agreement with reported measures of in vivo 5-HT1A activity, which were in part the basis of a recently proposed model for the 5-HT1A pharmacophore [J. Med. Chem. 34: 497-510 (1991)].     

The Na<Superscript>+</Superscript>/Cl<Superscript>−</Superscript>-Coupled,Broad-Specific,Amino Acid Transporter SLC6A14 (ATB<Superscript>0,+</Superscript>): Emerging Roles in Multiple Diseases and Therapeutic Potential for Treatment and Diagnosis

Amino acids are essential building blocks of all mammalian cells, and amino acid transporters play a vital role in transporting them into cells and their further distribution among the various cellular compartments. There are ~?430 known transporters in the solute-linked carrier (SLC) gene family, divided into 52 distinct families. Eleven of these gene families contain one or more amino acid transporters. These transporters differ significantly from each other in terms of substrate specificity, ion dependence, and energetics. Given the variety of roles they fulfill in human physiology, it is not surprising that a number of diseases are associated with the malfunction of these transporters. In particular, as amino acids are critical for cell growth, survival, and proliferation, the role of amino acid transporters in cancer is gaining increasing attention in recent years. The present review primarily focuses on one particular amino acid transporter, SLC6A14 (also known as ATB^0,+), with regard to its relevance to specific diseases, including cancer, and the molecular mechanisms underlying the disease-related alterations in the expression of the transporter. Furthermore, the review highlights the possible utility of this transporter in drug delivery and also its therapeutic potential for the treatment and diagnosis of cancer.     

Hydrolysis of irinotecan and its oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases CES1A1, CES2, and a newly expressed carboxylesterase isoenzyme, CES3.

Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min(-1) microM(-1) among the three carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min(-1) microM(-1). APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 x 10(-3) min(-1) microM(-1) for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 > CES3, for all three substrates.     

Efficacy of antipsychotic agents at human 5-HT(1A) receptors determined by [3H]WAY100,635 binding affinity ratios: relationship to efficacy for G-protein activation.

5-HT(1A) receptors are implicated in the aetiology of schizophrenia. Herein, the influence of 15 antipsychotics on the binding of the selective 'neutral' antagonist, [3H]WAY100,635 ([3H]N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclo-hexanecarboxamide), was examined at human 5-HT(1A) receptors expressed in Chinese Hamster Ovary cells. In competition binding experiments, 5-HT displayed biphasic isotherms which were shifted to the right in the presence of the G-protein uncoupling agent, GTPgammaS (100 microM). In analogy, the isotherms of ziprasidone, quetiapine and S16924 (((R-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]-pyrrolidin-3yl]-1-(4-fluoro-phenyl)-ethanone), were displaced to the right by GTPgammaS, consistent with agonist actions. Binding of several other antipsychotics, such as ocaperidone, olanzapine and risperidone, was little influenced by GTPgammaS. Isotherms of the neuroleptics, haloperidol, chlorpromazine and thioridazine were shifted to the left in the presence of GTPgammaS, suggesting inverse agonist properties. For most ligands, the magnitude of affinity changes induced by GTPgammaS (alteration in pK(i) values) correlated well with their previously determined efficacies in [35S]GTPgammaS binding studies [Eur. J. Pharmacol. 355 (1998) 245]. In contrast, the affinity of the 'atypical' antipsychotic agent, clozapine, which is a known partial agonist at 5-HT(1A) receptors, was less influenced by GTPgammaS. When the ratio of high-/low-affinity values was plotted against efficacy, hyperbolic isotherms were obtained, consistent with a modified ternary complex model which assumes that receptors can adopt active conformations in the absence of agonist. In conclusion, modulation of [3H]-WAY100,635 binding by GTPgammaS differentiated agonist vs. inverse agonist properties of antipsychotics at 5-HT(1A) receptors. These may contribute to differing profiles of antipsychotic activity.