Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3

1.?Organic cation transporters (OCTs) play an important role in drug safety and efficacy. Protoberberine alkaloids are ubiquitous organic cations or weak bases with remarkable biological actives. This study was to elucidate the potential interaction of alkaloids (coptisine, jatrorrhizine, epiberberine, berberrubine, palmatine and corydaline) with OCTs using Madin–Darby canine kidney (MDCK) cells stably expressing human OCT1, OCT2 and OCT3.

2.?All the tested alkaloids significantly inhibited the uptake of MPP⁺, a model OCT substrate, in MDCK-hOCTs cells with the IC₅₀ of 0.931–9.65?μM. Additionally, coptisine, jatrorrhizine and epiberberine were substrates of all the hOCTs with the K_m of 0.273–5.80?μM, whereas berberrubine was a substrate for hOCT1 and hOCT2, but not for hOCT3, the K_m values were 1.27 and 1.66?μM, respectively. The transport capacity of coptisine in MDCK cells expressing the variants of hOCT1-P341L or hOCT2-A270S was significantly higher than that in wild-type (WT) cells with the Cl_int (V_max/K_m) of 379?±?7.4 and 433?±?5.7?μl/mg protein/min, respectively.

3.?The above data indicate that the tested alkaloids are potent inhibitors, and coptisine, jatrorrhizine, epiberberine and berberrubine are substrates of hOCT1, hOCT2 and/or hOCT3 with high affinity. In addition, the variants (OCT1-P341L and OCT2-A270S) possess higher transport capacity to coptisine than WT hOCTs.     

Epigallocatechin-3-gallate decreases the transport and metabolism of simvastatin in rats

1.?This study aimed to investigate the potential impact of epigallocatechin-3-gallate (EGCG) on the pharmacokinetic behaviors of simvastatin and its metabolite simvastatin acid and explored the possible role of metabolizing enzymes and transporters of this food–drug interaction.

2.?Female SD rats were intravenously administered with EGCG (5?mg/kg), ketoconazole (10?mg/kg) and rifampin (10?mg/kg), followed by intravenous administration of 2?mg/kg simvastatin. In vitro, the effects of EGCG on Cytochrome P450 enzymes (CYP450) and organic anion transporting polypeptides (OATPs) were studied using human hepatic microsomes and human embryonic kidney 293 (HEK293) cells overexpressing OATP1B1 or OATP1B3. The results showed that areas under concentration–time (AUC) curves of simvastatin and simvastatin acid increased by 2.21- and 1.4-fold while the clearance was reduced by 2.29- and 1.4-fold, respectively, when co-administered with EGCG. In vitro experiments suggested the inhibitory effect of EGCG on CYP enzymes (IC₅₀: 18.37?±?1.36?μM, 26.08?±?1.51?μM for simvastatin and simvastatin acid, respectively). Simvastatin transport by OATP1B1 and OATP1B3 was also inhibited by EGCG (IC₅₀: 8.68?±?1.27?μM and 22.67?±?1.42?μM, respectively).

3.?The presently reported novel food–drug interaction between EGCG and simvastatin involves the inhibition of not only CYP450 enzymes but also OATPs by EGCG.     

Interactions of the active components of Punica granatum (pomegranate) with the essential renal and hepatic human Solute Carrier transporters

Context: Solute carrier transporters (SLCs) are membrane proteins responsible for cellular influx of various substances including many pharmaceutical agents; therefore, they largely impact on drug disposition and elimination in body. Punica granatum Linnaeus (Lythraceae), pomegranate, is a fruit with antidiabetic potential. Oleanolic acid (OA), ursolic acid (UA), and gallic acid (GA) are the major bioactive components of pomegranate. Co-administration of these compounds with other drugs could result in altered drug pharmacokinetics, possibly due to competing for transporter proteins.

Objective: We investigated the interactions of these three compounds with the essential hepatic and renal SLC transporters.

Materials and methods: Uptake of radiolabeled transporter model substrates was assessed in HEK293 cells over-expressing SLC transporters including the organic anion transporters (OATs), organic anion transporting polypeptides (OATPs) and organic cation transporters (OCTs), in the presence or absence of 10.0?µM UA, OA, or GA. Their IC₅₀ values on specific SLC transporters were also evaluated using varying concentrations of the particular compound (ranging from 0.10?nM to 80.0?µM).

Results: Our results demonstrated UA could significantly inhibit OAT3 and OATP2B1 uptake (IC₅₀: 18.9?±?8.20?µM and 11.0?±?5.00?µM, respectively) and GA has a pronounced inhibitory effect on OATP1B3 uptake (IC₅₀: 1.60?±?0.60?μM).

Discussion and conclusion: Our study reports the interactions of OA, UA, and GA with the essential SLC transporters. This information may contribute to elucidating the drug–drug/herb interactions involved with these three compounds and form the basis of therapeutic optimization when drugs are co-administered.     

The inhibitory effects of camptothecin (CPT) and its derivatives on the substrate uptakes mediated by human solute carrier transporters (SLCs)

1.?Camptothecin (CPT) and its derivatives are potent candidate compounds in treating cancers. However, their clinical applications are largely restricted by severe toxicities.

2.?The solute carrier transporters (SLCs), particularly the organic anion transporting polypeptides and organic anion/cation transporters (OATs/OCTs) are widely expressed in human key organs and responsible for the cellular influx of many substances including endogenous substrates and many clinically important drugs. Drug–drug interactions through SLCs often result in unsatisfied therapeutic outcomes and/or unexpected toxicities.

3.?This study investigated the inhibitory effects of CPT and its eight derivatives on the cellular uptake of specific substrates mediated by the essential SLCs in over-expressing Human embryonic kidney 293 cells.

4.?Our data revealed that CPT, 10-hydroxycamptothecin (HCPT), 10-methoxycamptothecin (MCPT) and 9-nitrocamptothecin (9NC) significantly inhibit the uptake activity of OAT3. 9NC also inhibited the substrate transport mediated by OAT1. The substrate uptakes of OAT1, OCTN1 and OCTN2 were significantly decreased in the presence of CZ112, while CPT-11 potently down-regulated the transport activity of OCT1 and OCT3.

5.?In summary, our study demonstrated that CPT and its eight derivatives selectively inhibit the substrate uptakes mediated by the essential SLCs. This information contributes to understanding the localized toxicity of CPTs and provides novel molecular targets for the therapeutic optimization of CPTs in the future.     

Interaction of green tea catechins with renal organic cation transporter 2

1.?Green tea extract (GTE) and EGCG have previously shown to increase the uptake of MPP⁺ into Caco-2 cells. However, whether GTE and its derivatives interact with renal basolateral organic cation transporter 2 (Oct2) which plays a crucial role for cationic clearance remains unknown. Thus, this study assessed the potential of drug-green tea (GT) catechins and its derivatives interactions with rat Oct2 using renal cortical slices and S2 stably expressing rat Oct2 (S2rOct2).

2.?Both GTE and ECG inhibited MPP⁺ uptake in renal slices in a concentration-dependent manner (IC₅₀?=?2.71?±?0.360?mg/ml and 0.87?±?0.151?mM), and this inhibitory effect was reversible. Inhibition of [³H]MPP⁺ transport in S2rOct2 by either GTE or ECG (IC₅₀?=?1.90?±?0.087?mg/ml and 1.67?±?0.088?mM) was also observed.

3.?The weak and reversible interactions of GTE and ECG with rOct2 indicate that consumption of GT beverages could not interfere with cationic drugs secreted via renal OCT2 in humans. However, the rise of therapeutic use of GTE and ECG might have to take into account the significant possibility of adverse drug–green tea catechins interactions which could alter renal organic cation drug clearance.     

Potent inhibition of human organic cation transporter 2 (hOCT2) by β-carboline alkaloids

1.?Beta-carbolines are indole alkaloids with a wide range of pharmacological and toxicological activities. Beta-carbolines are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), a known substrate of organic cation transporters (OCTs). The goal of this study is to determine the interaction of β-carbolines with human OCT1, 2, and 3 (SLC22A1-3).

2.?Dose-dependent inhibition studies were performed for five commercially available β-carbolines using a fluorescent substrate assay in HEK293 cells stably expressing hOCT1-3. The substrate potential was evaluated by uptake assays and the impact of active transport on cellular toxicity examined.

3.?All tested β-carbolines potently inhibited hOCT2 with IC₅₀ values in the sub- or low micromolar range. Harmaline is the most potent hOCT2 inhibitor (IC_50?=_?0.50?±?0.08?μM). hOCT1 and hOCT3 are less sensitive to β-carboline inhibition. Harmaline, norharmanium, and 2,9-dimethyl-4,9-dihydro-3H-β-carbolinium accumulated 2- to 7-fold higher in cells expressing hOCT1-3. HEK293 cells expressing hOCT1-3 were 6.5- to 13-fold more sensitive to harmane and norharmanium toxicity.

4.?Our data support a significant role of hOCT1-3 in tissue uptake and disposition of β-carbolines. Importantly, the potent inhibition of hOCT2 by β-carbolines also raises the concern of potential drug interactions between naturally occurring bioactive alkaloids and drugs eliminated by hOCT2.     

Investigation of the role of organic cation transporter 2 (OCT2) in the renal transport of guanfacine,a selective α2A-adrenoreceptor agonist

Abstract

1.?Guanfacine is a selective α_2A-adrenoreceptor agonist primarily excreted as its unchanged form through urine in human. This study was to investigate the involvement of organic cation transporter 2 (OCT2) in the renal tubular secretion of guanfacine.

2.?Transport of guanfacine was characterized using human embryonic kidney (HEK293) cells expressing human OCT2 (hOCT2). The inhibitory effect of cimetidine on guanfacine uptake was also examined. In addition, in vivo pharmacokinetic study was conducted in rats to assess the effects of cimetidine on the pharmacokinetics of guanfacine.

3.?The accumulation of guanfacine in hOCT2-transfected HEK293 cells was both time- and concentration-dependent, and markedly higher than that in mock cells. The apparent K_m and V_max values of guanfacine uptake by hOCT2 were 96.19?±?7.49?μM and 13.03?±?0.49?nmol/mg protein/min, respectively. Guanfacine transport mediated by hOCT2 was significantly inhibited by a typical OCT2 inhibitor cimetidine with an IC₅₀ value of 93.82?±?1.13?μM. Co-administration of cimetidine significantly decreased the plasma clearance (CL_p) as well as the renal clearance (CL_r) of guanfacine in rats in a dose-dependent manner, resulting in a noticeable increase in the systemic exposure of guanfacine.

4.?These results indicated that OCT2 may be involved in the renal disposition of guanfacine.     

Enantiospecific effects of chiral drugs on cytochrome P450 inhibition in vitro

1.?The aim of this work was to examine the differences in the inhibitory potency of individual enantiomers and racemic mixtures of selected chiral drugs on human liver microsomal cytochromes P450.

2.?The interaction of enantiomeric forms of six drugs (tamsulosin, tolterodine, citalopram, modafinil, zopiclone, ketoconazole) with nine cytochromes P450 (CYP3A4, CYP2E1, CYP2D6, CYP2C19, CYP2C9, CYP2C8, CYP2B6, CYP2A6, CYP1A2) was examined. HPLC methods were used to estimate the extent of the inhibition of specific activity in vitro.

3.?Tamsulosin (TAM) and tolterodine (TOL) inhibited CYP3A4 activity with an enantiospecific pattern. The inhibition of CYP3A4 activity differed for R-TAM (K_i 2.88?±?0.12?µM) and S-TAM (K_i 14.22?±?0.53?µM) as well as for S-TOL (K_i 1.71?±?0.03?µM) and R-TOL (K_i 4.78?±?0.17?µM). Also, the inhibition of CYP2C19 by ketoconazole (KET) cis-enantiomers exhibited enantioselective behavior: the (+)-KET (IC₅₀ 23.64?±?6.25?µM) was more potent than (?)-KET (IC₅₀ 66.12?±?12.6?µM). The inhibition of CYP2C19 by modafinil (MOD) enantiomers (R-MOD IC₅₀?=?51.79?±?8.58?µM, S-MOD IC₅₀?=?48.62?±?9.74?µM) and the inhibition of CYP2D6 by citalopram (CIT) enantiomers (R-CIT IC₅₀?=?68.17?±?5.70?µM, S-CIT IC₅₀?=?62.63?±?7.89?µM) was not enantiospecific.

4.?Although enantiospecific interactions were found (TAM, TOL, KET), they are probably not clinically relevant as the plasma levels are generally lower than the drug concentration needed for prominent inhibition (at least 50% of CYP activity).     

Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III

1.?Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of organic acids into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3.

2.?This hypothesis was tested using cells transfected with OATP1B1 and OATP1B3. OATP1B-mediated transport of coproporphyrin was time-dependent and concentration-dependent. OATP1B1-mediated transport of coproporphyrins I and III (K_m?=?0.13 and 0.22?µM, respectively), as did OATP1B3 (K_m?=?3.25 and 4.61?µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifampicin.

3.?The specificity of coproporphyrin transport was also investigated where OATP2B1 demonstrated meaningful transport of coproporphyrin III (K_m?=?0.31?µM), while OCT1, OCT2, OAT1, OAT3 and NTCP were negative for coproporphyrin transport.

4.?The identification of coproporphyrins as OATP substrates in vitro more clearly defines the role of OATPs in the hepatic disposition and renal excretion of coproporphyrins I and III and provides compelling evidence for future in vivo exploration of coproporphyrins as biomarkers of OATP activity.     

In vitro inhibition of UGT1A3, UGT1A4 by ursolic acid and oleanolic acid and drug–drug interaction risk prediction

1.?Ursolic acid (UA) and oleanolic acid (OA) may have important activity relevant to health and disease prevention. Thus, we studied the activity of UA and OA on UDP-glucuronosyltransferases (UGTs) and used trifluoperazine as a probe substrate to test UGT1A4 activity. Recombinant UGT-catalyzed 4-methylumbelliferone (4-MU) glucuronidation was used as a probe reaction for other UGT isoforms.

2.?UA and OA inhibited UGT1A3 and UGT1A4 activity but did not inhibit other tested UGT isoforms.

3.?UA-mediated inhibition of UGT1A3 catalyzed 4-MU-β-d-glucuronidation was via competitive inhibition (IC₅₀ 0.391?±?0.013?μM; K_i 0.185?±?0.015?μM). UA also competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 2.651?±?0.201?μM; K_i 1.334?±?0.146?μM).

4.?OA offered mixed inhibition of UGT1A3-mediated 4-MU-β-d-glucuronidation (IC₅₀ 0.336?±?0.013?μM; K_i 0.176?±?0.007?μM) and competitively inhibited UGT1A4-mediated trifluoperazine-N-glucuronidation (IC₅₀ 5.468?±?0.697?μM; K_i 6.298?±?0.891?μM).

5.?Co-administering OA or UA with drugs or products that are substrates of UGT1A3 or UGT1A4 may produce drug-mediated side effects.     

Genetic variants of organic cation transporter 2 (OCT2) significantly reduce metformin uptake in oocytes

1.?The authors sought to evaluate the contribution of organic cation transporters (OCTs) to the renal tubular transport of metformin using LLC-PK1 cells as an in vitro model for the renal proximal tubule, and to investigate the effects of three non-synonymous genetic variants of OCT2 on the transport activity of metformin in vitro using an oocyte over-expression system.

2.?The basolateral-to-apical transport of metformin was significantly greater than the apical-to-basolateral transport and showed concentration dependency with the kinetic parameters: maximum transport rate (V_max), 922 pmol min^?1 per 5 × 10⁵ cells; Michaelis–Menten constant (K_m), 393 µM; intrinsic clearance (CL_int), 2.35 µl min^?1 per 5 × 10⁵ cells; and diffusion constant (K_d), 0.33 µl min^?1 per 5 × 10⁵ cells. The basolateral-to-apical transport of metformin was inhibited by phenoxybenzamine, an inhibitor of OCTs, but not by cyclosporine A, MK571, or fumitremorgin C, which are inhibitors of P-glycoprotein, multidrug resistance proteins (MRPs), and breast cancer resistance protein (BCRP), respectively, suggesting that OCTs play a role in renal tubular secretion of metformin.

3.?Metformin uptake was much greater in oocytes expressing OCT2-wild type (OCT2-WT) than OCT1-WT compared with uptake in water-injected oocytes. Uptake was significantly decreased in oocytes expressing OCT2-T199I, -T201M, and -A270S compared with that in OCT2-WT, suggesting that metformin is a better substrate for OCT2 than for OCT1 and that the amino acid-substituted variants of OCT2 cause a functional decrease in metformin uptake.

4.?In conclusion, the genetic variants of OCT2 (OCT2-T199I, -T201M, and -A270S) decreased the transport activity of metformin and thus may contribute to the inter-individual variation in metformin disposition as OCT2 plays a pivotal role in renal excretion, the major disposition route of metformin.     

Interactions of organophosphorus pesticides with solute carrier (SLC) drug transporters

1.?Organophosphorus pesticides (OPs) are known to interact with human ATP-binding cassette drug efflux pumps. The present study was designed to determine whether they can also target activities of human solute carrier (SLC) drug transporters.

2.?The interactions of 13 OPs with SLC transporters involved in drug disposition, such as organic cation transporters (OCTs), multidrug and toxin extrusion proteins (MATEs), organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs), were mainly investigated using transporter-overexpressing cell clones and fluorescent or radiolabeled reference substrates.

3.?With a cut-off value of at least 50% modulation of transporter activity by 100?µM OPs, OAT1 and MATE2-K were not impacted, whereas OATP1B1 and MATE1 were inhibited by two and three OPs, respectively. OAT3 activity was similarly blocked by three OPs, and was additionally stimulated by one OP. Five OPs cis-stimulated OATP2B1 activity. Both OCT1 and OCT2 were inhibited by the same eight OPs, including fenamiphos and phosmet, with IC₅₀ values however in the 3–30?µM range, likely not relevant to environmental exposure.

4.?These data demonstrated that various OPs inhibit SLC drug transporter activities, especially those of OCT1 and OCT2, but only when used at high concentrations not expected to occur in environmentally-exposed humans.     

Interactions of Stevioside and Steviol with Renal Organic Anion Transporters in S2 Cells and Mouse Renal Cortical Slices

Purpose Our previous studies have shown that both stevioside and steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with organic anion transport system. The current study examined the direct interactions of stevioside and steviol with specific organic anion transporters.Methods S2 cells expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion transport.Results Stevioside at 0.5–1 mM showed no interaction with any OAT. In contrast, steviol markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC₅₀ for hOAT1 (11.4 M) and hOAT3 (36.5 M) similar to that of probenecid, whereas IC₅₀ for hOAT2 (1000 M) and hOAT4 (285 M) was much higher. Results obtained in mouse renal cortical slices were very similar; that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone sulfate (ES) transport.Conclusions Stevioside has no interaction with human or mouse OATs. In contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter renal drug clearance.     

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1.?In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.

2.?Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC₅₀ values of 20.3?μM and 27.1?μM) and OCT1 (IC₅₀ 37.9?μM), but limited inhibition of OATP1B3 (25% at 100?μM); inhibition of the renal uptake transporters OCT2 (IC₅₀ 19.9?μM) and OAT3 (IC₅₀ 18.4?μM), but limited inhibition of OAT1 (13.5% at 100?μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC₅₀s 5.50?μM and 47.1?μM, respectively); inhibition of the efflux transporter MDR1 (IC₅₀ 76.0?μM), but limited inhibition of BCRP (47% at 100?μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.

3.?Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.     

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin,an SGLT2 inhibitor

1.?We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively.

2.?Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC₅₀ value of 58.3?μM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72?hrs weakly induced CYP3A4 at a concentration of 10?μM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1–10?μM.

3.?An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC₅₀ value of 93.1?μM, but those for other transporters are greater than 100?μM.

4.?Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.     

Diorganotin (IV) derivative of 2-thiophene acetic acid: characterizations and biological activities of {[n-Bu2SnO2C–CH2–C4H3S]2O}2

New organotin (IV) derivatives of thiophene acetic acid have been prepared and characterized by IR, ¹H- and ¹³C-NMR spectroscopic techniques. A single crystal of {[n-Bu₂SnO₂C–CH₂–C₄H₃S]₂O}₂ has been synthesized and its cell parameters were measured. It crystallizes in the monoclinic system (P2₁) [a?=?15.1337(7), b?=?12.2587(5), c?=?18.8766(9), and β?=?105.811(5)°]. The compound showed selective inhibitory effect against β-glucuronidase enzyme (IC₅₀ 3.1?±?0.1?μM), which is more potent than our standard, d-saccharic acid 1,4-lactone (IC₅₀ 48.4?±?1.3?μM). Also, it exhibited immunomodulatory activity, and cytotoxicity against PC-3 cell line (IC₅₀?=?16.9?±?1.3?μM, and 1.2?±?0.1?μM, respectively), which are close to the standards ibuprofen (IC₅₀ 11.8?±?1.8?μM), and doxorubicin (IC₅₀ 0.9?±?0.1?μM), respectively. This compound did not show any significant inhibition for other biological test such as α-chymotrypsin, urease, phosphodiesterase enzymes, and antiglycation activity.     

Predominant contributions of carboxylesterase 1 and 2 in hydrolysis of anordrin in humans

1.?Anordrin (2α, 17α-diethynyl-A-nor-5α-androstane-2β, 17β-diol diproprionate) is post-coital contraceptive drug that is on the market in China for more than 30 years. This study aims to elucidate enzymes involved in anordrin hydrolysis, and to evaluate the significant role of carboxylesterases in anordrin hydrolysis in humans.

2.?Human liver and intestinal microsomes, recombinant human carboxylesterase were selected as enzyme sources. In human liver microsomes, intrinsic clearance was 684?±?83?μL/min/mg protein, which was considerably higher than the value of intestine microsomes (94.6?±?13.3?μL/min/mg protein). Carboxylesterase (CES) 1 has more contribution than CES2 in human liver.

3.?Inhibition studies were performed using representative esterase inhibitors to confirm esterase isoforms involved in anordrin hydrolysis. Simvastatin strongly inhibited hydrolytic process of anordrin in liver and intestine microsomes, with IC₅₀ values of 10.9?±?0.1 and 6.94?±?0.03?μM, respectively.

4.?The present study investigated for the first time hydrolytic enzyme phenotypes of anordrin. Anordrin is predominantly catalyzed by CES1 and CES2 to generate the main active metabolite, anordiol. Moreover, anordrin and its metabolite anordiol can be altered by esterase inhibitors, such as simvastatin, upon exposure in vivo.     

Xanthine oxidase/tyrosinase inhibiting,antioxidant, and antifungal oxindole alkaloids from Isatis costata

Phytochemical investigations on the ethyl acetate soluble fraction of the whole plant of Isatis costata Linn. (Brassicaseae) led to the isolation of the oxindole alkaloids costinones A (1), B (2), isatinones A (3), B (4), indirubin (5), and trisindoline (6). Compounds 1–6 displayed significant to moderate inhibition against xanthine oxidase enzyme with IC₅₀ values ranging from 90.3?±?0.06 to 179.6?±?0.04 µM, whereas the standard inhibitor of xanthine oxidase (allopurinol) had an IC₅₀ value of 7.4?±?0.07 µM. Compounds 1 (IC₅₀ 7.21?±?0.05 µM), 2 (IC₅₀ 9.40?±?0.03 µM), 3 (IC₅₀ 11.51?±?0.07 µM), 4 (IC₅₀ 12.53?±?0.06 µM), 5 (IC₅₀ 14.29?±?0.09 µM), and 6 (IC₅₀ 17.34?±?0.04 µM) exhibited pronounced activities when compared with the standard tyrosinase inhibitor l-mimosine (IC₅₀ 3.70?±?0.03 µM), along with DPPH radical scavenging activity with IC₅₀ 226, 270, 300, 320, 401, and 431 µM, respectively. The crude extract and compounds 1, 2, 5, and 6 showed significant antifungal activity against Trichophyton schoen leinii, Aspergillus niger, Candida albicans, Trichophyton simii, and Macrophomina phaseolina.     

Jatrorrhizine reduces 5-HT and NE uptake via inhibition of uptake-2 transporters and produces antidepressant-like action in mice

Abstract

1. Jatrorrhizine is an active ingredient found in various traditional Chinese medicinal plants. Based on our previous finding that jatrorrhizine was a potent inhibitor of OCT2 and OCT3, the aim of the present study was to explore whether jatrorrhizine has an antidepressant-like action action via inhibition of uptake-2 transporters.

2. In vitro uptake tests showed that jatrorrhizine strongly inhibited PMAT-mediated MPP⁺ uptake with an IC₅₀ value of 1.05?μM and reduced 5-HT and NE uptake mediated by hOCT2, hOCT3 and hPMAT with IC₅₀ values of 0.1–1?μM (for OCT2 and OCT3) and 1–10?μM (for PMAT).

3. In mouse synaptosomes, jatrorrhizine suppressed 5-HT and NE uptake in a concentration dependently manner, where the role of uptake-2 inhibition is significant.

4. The antidepressant-like action of jatrorrhizine was evaluated by mouse tail suspension test (TST). The TST showed that one week of jatrorrhizine (5, 10 and 20?mg/kg, i.p.) or venlafaxine (20?mg/kg, i.g.) can significantly reduce the duration of immobility when compared with vehicle control group.

5. The concentration of jatrorrhizine shows a dose-dependent increase in brain tissues.

6. Our study suggested that jatrorrhizine might be used as an antidepressant agent via inhibition of uptake-2 transporters.     

The Inhibitory Effects of the Bioactive Components Isolated from Scutellaria Baicalensis on the Cellular Uptake Mediated by the Essential Solute Carrier Transporters

Solute carrier transporters (SLCs), in particular the organic anion transporters (OATs), OAT polypeptides (OATPs), and organic cation transporters (OCTs/OCTNs), are the important membrane proteins responsible for the cellular influx of various drugs. Baicalein (BA), baicalin (BG), and wogonin (WG) are the three major bioactive components of Scutellaria baicalensis. In this study, we evaluated the inhibitory effects of BA, BG, and WG on the cellular uptake of specific substrates mediated by the essential SLCs in human embryonic kidney-293 cells. Our data demonstrated that BA and WG significantly inhibit the OAT1-, OAT3-, and OATP1B3-mediated uptake; BG effectively reduces the influx of substrates of OAT3, OAT4, OATP1B3, and OATP2B1; WG is a potent inhibitor of OCT3. Our further kinetic analysis derived the IC₅₀ values of these compounds with pronounced inhibitory effects on SLCs, particularly the inhibitions of WG on OAT1 and OCT3 and that of BA and WG on OAT3. Our study comprehensively evaluated the inhibitory effects of three bioactive components of Scutellaria baicalensis on the uptake of specific substrates mediated by the essential SLC transporters, which suggested that precautions will be needed when coadministrating drugs with Scutellaria baicalensis so as to prevent the unfavorable drug-drug/herb interactions in human. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:4205-421 1, 2013