Publications by authors named "Huidi Jiang"

89 Publications

L-tetrahydropalmatine reduces oxaliplatin accumulation in the dorsal root ganglion and mitochondria through selectively inhibiting the transporter-mediated uptake thereby attenuates peripheral neurotoxicity.

Toxicology 2021 Jul 9;459:152853. Epub 2021 Jul 9.

Laboratory of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, PR China. Electronic address:

Oxaliplatin (OXA) is a third-generation platinum drug; however, its application is greatly limited due to the severe peripheral neurotoxicity. This study aims to confirm the transport mechanism of OXA and to explore whether L-tetrahydropalmatine (L-THP) would alleviate OXA-induced peripheral neurotoxicity by selectively inhibiting these uptake transporters in vitro and in vivo. Our results revealed that organic cation transporter 2 (OCT2), organic cation/carnitine transporter 1 (OCTN1) and organic cation/carnitine transporter 2 (OCTN2) were involved in the uptake of OXA in dorsal root ganglion (DRG) neurons and mitochondria, respectively. L-THP (1-100 μM) reduced OXA (40 μM) induced cytotoxicity in MDCK-hOCT2 (Madin-Darby canine kidney, MDCK), MDCK-hOCTN1, MDCK-hOCTN2, and rat primary DRG cells, and decreased the accumulation of OXA in above cells and rat DRG mitochondria, but did not affect its efflux from MDCK-hMRP2 cells. Furthermore, Co-administration of L-THP (5-20 mg/kg for mice, 10-40 mg/kg for rats; twice a week, iv or ig) attenuated OXA (8 mg/kg for mice, 4 mg/kg for rats; twice a week, iv) induced peripheral neurotoxicity and reduced the platinum concentration in the DRG. Whereas, L-THP (1-100 μM for cells; 10-20 mg/kg for mice) did not impair the antitumour efficacy of OXA (40 μM for cells; 8 mg/kg for mice) in HT29 tumour-bearing nude mice nor in tumour cells (HT29 and SW620 cells). In conclusion, OCT2, OCTN1 and OCTN2 contribute to OXA uptake in the DRG and mitochondria. L-THP attenuates OXA-induced peripheral neurotoxicity via inhibiting OXA uptake but without impairing the antitumour efficacy of OXA. L-THP is a potential candidate drug to attenuate OXA-induced peripheral neurotoxicity.
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http://dx.doi.org/10.1016/j.tox.2021.152853DOI Listing
July 2021

Gut inflammation exacerbates high-fat diet induced steatosis by suppressing VLDL-TG secretion through HNF4α pathway.

Free Radic Biol Med 2021 Jun 26;172:459-469. Epub 2021 Jun 26.

Laboratory of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address:

Nonalcoholic fatty liver disease (NAFLD) is increasingly identified in inflammatory bowel disease (IBD) patients with unclear etiology. In the current study we assessed the contribution of colonic inflammation to NAFLD development and the underlying mechanism in a mouse model for IBD. Our results showed that dextran sulfate sodium (DSS)-induced gut colitis directly led to hepatic inflammation, injury and further exacerbated hepatic steatosis caused by high fat diet (HF) feeding. The essential genes assessment, hepatic metabolic analysis and triglyceride-rich very low-density lipoprotein (VLDL-TG) secretion assays revealed a higher β-oxidation of fatty acids (FAs) but impaired VLDL-TG secretion in liver of DSS-treated mice. Disruption of the intestinal barrier by DSS promoted liver inflammation, which strongly suppressed hepatic VLDL-TG secretion and further aggravated HF-induced VLDL-TG secretion impairment through down-regulation of apolipoprotein B (APOB), hence promoting the storage of triglycerides (TG) in the liver. Inflammation induced by mixed proinflammatory cytokines or LPS obviously inhibited the expression of microsomal triglyceride transfer protein (MTP) and APOB expression and subsequently increased TG content via the suppression of HNF4α in mouse primary hepatocytes. In addition, the downregulation of MTP and APOB by proinflammatory cytokines was also rescued through activating Hnf4α by cortisol. Altogether, our results demonstrated that chronic inflammation exacerbated hepatic steatosis by inhibiting the secreting of hepatic VLDL-TG through HNF4α pathway, suggesting that restoring hepatic VLDL-TG secretion may be a novel strategy for treatment of NAFLD in IBD.
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http://dx.doi.org/10.1016/j.freeradbiomed.2021.06.027DOI Listing
June 2021

Downregulation of glucose-6-phosphatase expression contributes to fluoxetine-induced hepatic steatosis.

J Appl Toxicol 2021 Aug 12;41(8):1232-1240. Epub 2020 Nov 12.

Institute of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.

Fluoxetine is a first-line selective serotonin reuptake inhibitor widely applied for the treatment of depression; however, it induces abnormal hepatic lipid metabolism. Considering decreased expression or function of glucose-6-phosphatase (G6Pase), a key enzyme in gluconeogenesis, or the upregulation of fatty acid uptake, causes hepatic lipid accumulation. The aim of this study was to elucidate whether G6Pase regulation and fatty acid uptake alteration contribute to fluoxetine-induced abnormal hepatic lipid metabolism. Our study revealed that 8-week oral administration of fluoxetine dose-dependently increased hepatic triglyceride, causing hepatic steatosis. Concomitantly, the expression of G6Pase in mouse livers and primary mouse hepatocytes (PMHs) was downregulated in a concentration-dependent manner. Furthermore, fluoxetine increased the concentrations of glucose-6-phosphate (G6Pase substrate) and acetyl CoA (the substrate for de novo lipogenesis) in mouse livers. Additionally, fluoxetine also induced lipid accumulation and downregulated G6Pase expression in HepG2 cells. However, the uptake of green fluorescent fatty acid (BODIPY™ FL C16) in PMHs was not changed after fluoxetine treatment, indicating that fluoxetine-induced hepatic steatosis was not associated with fatty acid uptake alteration. In conclusion, fluoxetine downregulated hepatic G6Pase expression, subsequently enhanced the transformation of glucose to lipid, and ultimately resulted in hepatic steatosis, but with no impact on fatty acid uptake.
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http://dx.doi.org/10.1002/jat.4109DOI Listing
August 2021

Effects of rhein and Rheum palmatum L. extract on the pharmacokinetics and tissue distribution of aristolochic acid I and its demethylated metabolite in rats.

J Ethnopharmacol 2021 Mar 1;267:113537. Epub 2020 Nov 1.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China. Electronic address:

Ethnopharmacological Relevance: Aristolochic acid nephropathy (AAN) is a kidney disease caused by the administration of plants containing aristolochic acids (AAs). Aristolochic acid I (AAI) is the main toxic component in AAs. Organic anion transporters (OATs) 1 and 3 mediate the renal uptake of AAI, which is related to AAN. In our previous study, we found that anthraquinones derived from the herbal medicine Rheum palmatum L. (RP) inhibited both OAT1 and OAT3, with rhein exhibiting the greatest potency among the components.

Aim Of The Study: This study aimed to investigate the effects of rhein and RP extract on the pharmacokinetics and tissue distribution of AAI and its demethylated metabolite (8-hydroxy-aristolochic acid I [AAIa]) in rats.

Materials And Methods: Rhein and RP extract were used as OAT inhibitors, and AAI was used as the toxic substrate. The pharmacokinetics and tissue distribution of AAI and AAIa in rats following the intravenous injection of AAI (10 mg/kg) in the presence and absence of rhein (100 mg/kg) or RP extract (5 g crude drug/kg) were investigated.

Results: Co-administration with rhein increased AUC of AAI and AAIa by 39 and 44%, respectively. However, the renal level of AAI was decreased to 50, 42, and 58% of those in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively, and the renal level of AAIa was decreased to 58, 57, and 61% of the level in rats treated with AAI alone, respectively, at these time points. In the RP extract co-administration group, AAI and AAIa plasma exposure was not significantly increased, but renal accumulation of AAI was decreased to 63, 58, and 68% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively. In addition, renal accumulation of AAIa was decreased to 74, 70, and 70% of that in rats treated with AAI alone at 5, 10, and 20 min after treatment, respectively.

Conclusions: This study indicated that co-administration with rhein significantly increased the plasma exposure of AAI and AAIa while decreased their renal accumulation in rats. RP extract reduced the renal accumulation of AAI and AAIa, but have no significant effect on their plasma exposure levels in rats.
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http://dx.doi.org/10.1016/j.jep.2020.113537DOI Listing
March 2021

Organic Cation Transporter 1 and 3 Contribute to the High Accumulation of Dehydrocorydaline in the Heart.

Drug Metab Dispos 2020 10 28;48(10):1074-1083. Epub 2020 Jul 28.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China

Dehydrocorydaline (DHC), one of the main active components of , is an important remedy for the treatment of coronary heart disease. Our previous study revealed a higher unbound concentration of DHC in the heart than plasma of mice after oral administration of extract or DHC, but the underlying uptake mechanism remains unelucidated. In our investigations, we studied the transport mechanism of DHC in transgenic cells, primary neonatal rat cardiomyocytes, and animal experiments. Using quantitative real-time polymerase chain reaction and Western blotting, we found that uptake transporters expressed in the mouse heart include organic cation transporter 1/3 (OCT1/3) and carnitine/organic cation transporter 1/2 (OCTN1/2). The accumulation experiments in transfected cells showed that DHC was a substrate of OCT1 and OCT3, with of 11.29 ± 3.3 and 8.96 ± 3.7 μM, respectively, but not a substrate of OCTN1/2. Additionally, a higher efflux level (1.71-fold of MDCK-mock) of DHC was observed in MDCK-MDR1 cells than in MDCK-mock cells. Therefore, DHC is a weak substrate for MDR1. Studies using primary neonatal rat cardiomyocytes showed that OCT1/3 inhibitors (quinidine, decynium-22, and levo-tetrahydropalmatine) prevented the accumulation of DHC, whereas OCTN2 inhibitors (mildronate and l-carnitine) did not affect its accumulation. Moreover, the coadministration of OCT1/3 inhibitors (levo-tetrahydropalmatine, THP) decreased the concentration of DHC in the mouse heart. Based on these findings, DHC may be accumulated partly by OCT1/3 transporters and excreted by MDR1 in the heart. THP could alter the distribution of DHC in the mouse heart. SIGNIFICANCE STATEMENT: We reported the cardiac transport mechanism of dehydrocorydaline, highly distributed to the heart after oral administration of o extract or dehydrocorydaline only. Dehydrocorydaline (an OCT1/3 and MDR1 substrate) accumulation in primary cardiomyocytes may be related to the transport activity of OCT1/3. This ability, hampered by selective inhibitors (levo-tetrahydropalmatine, an inhibitor of OCT1/3), causes a nearly 40% reduction in exposure of the heart to dehydrocorydaline. These results suggest that OCT1/3 may contribute to the uptake of dehydrocorydaline in the heart.
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http://dx.doi.org/10.1124/dmd.120.000025DOI Listing
October 2020

Epigenetic Regulation of Differentially Expressed Drug-Metabolizing Enzymes in Cancer.

Drug Metab Dispos 2020 09 29;48(9):759-768. Epub 2020 Jun 29.

Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (J.W., L.Y., H.J., S.Z.) and Hangzhou Cancer Institution, Hangzhou Cancer Hospital, Hangzhou, China (X.Z.)

Drug metabolism is a biotransformation process of drugs, catalyzed by drug-metabolizing enzymes (DMEs), including phase I DMEs and phase II DMEs. The aberrant expression of DMEs occurs in the different stages of cancer. It can contribute to the development of cancer and lead to individual variations in drug response by affecting the metabolic process of carcinogen and anticancer drugs. Apart from genetic polymorphisms, which we know the most about, current evidence indicates that epigenetic regulation is also central to the expression of DMEs. This review summarizes differentially expressed DMEs in cancer and related epigenetic changes, including DNA methylation, histone modification, and noncoding RNAs. Exploring the epigenetic regulation of differentially expressed DMEs can provide a basis for implementing individualized and rationalized medication. Meanwhile, it can promote the development of new biomarkers and targets for the diagnosis, treatment, and prognosis of cancer. SIGNIFICANCE STATEMENT: This review summarizes the aberrant expression of DMEs in cancer and the related epigenetic regulation of differentially expressed DMEs. Exploring the epigenetic regulatory mechanism of DMEs in cancer can help us to understand the role of DMEs in cancer progression and chemoresistance. Also, it provides a basis for developing new biomarkers and targets for the diagnosis, treatment, and prognosis of cancer.
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http://dx.doi.org/10.1124/dmd.120.000008DOI Listing
September 2020

Downregulation of OCTN2 by cytokines plays an important role in the progression of inflammatory bowel disease.

Biochem Pharmacol 2020 08 21;178:114115. Epub 2020 Jun 21.

Laboratory of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address:

Inflammatory bowel diseases (IBD) are characterized by chronic relapsing disorders of the gastrointestinal tract. OCTN2 (SLC22A5) and its substrate l-carnitine (l-Car) play crucial roles in maintaining normal intestinal function. An aim of this study was to delineate the expression alteration of OCTN2 in IBD and its underlying mechanism. We also investigated the impact of OCTN2 on IBD progression and the possibility of improving IBD through OCTN2 regulation. Our results showed decreased OCTN2 expression levels and l-Car content in inflamed colon tissues of IBD patients and mice, which negatively correlated with the degree of colonic inflammation in IBD mice. Mixed proinflammatory cytokines TNF-α, IL-1β and IFNγ downregulated the expression of OCTN2 and subsequently reduced the l-Car content through PPARγ/RXRα pathways in FHC cells. OCTN2 silencing reduced the proliferation rate of the colon cells, whereas OCTN2 overexpression increased the proliferation rate. Furthermore, the ability of PPARγ agonist, luteolin, to increase OCTN2 expression resulted in the alleviation of colonic inflammatory responses. In conclusion, OCTN2 was downregulated in IBD by proinflammatory cytokines via the PPARγ/RXRα pathways, which reduced l-Car concentration and subsequently induced IBD deterioration. Upregulation of OCTN2 by the PPARγ agonist alleviated colonic inflammation. Our findings suggest that, OCTN2 may serve as a therapeutic target for IBD therapy.
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http://dx.doi.org/10.1016/j.bcp.2020.114115DOI Listing
August 2020

L-tetrahydropalmatine attenuates cisplatin-induced nephrotoxicity via selective inhibition of organic cation transporter 2 without impairing its antitumor efficacy.

Biochem Pharmacol 2020 07 8;177:114021. Epub 2020 May 8.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, PR China. Electronic address:

Cisplatin is a first-line chemotherapeutic agent that is widely used for treatment of various solid tumors. However, cisplatin-induced adverse effects, particularly severe nephrotoxicity, preclude its application. In this study, we showed that L-tetrahydropalmatine (L-THP) could selectively inhibit organic cation transporter 2 (OCT2), which plays a crucial role in renal cisplatin uptake from the circulation. Additionally, we demonstrated that L-THP attenuated cisplatin-induced toxicity in mouse primary renal tubular cells. Subsequently, we verified that L-THP reduced the renal accumulation of cisplatin and alleviated cisplatin-induced renal injury in healthy and tumor-bearing nude mice. In healthy mice, co-treatment of L-THP at 5-40 mg/kg reduced cisplatin renal accumulation to 75.0%-49.9% of that in cisplatin alone group (10 mg/kg), and alleviated cisplatin-induced nephrotoxicity. Additionally, it did not alter Pt concentration in the tumor tissue and did not impair its antitumor efficacy in tumor bearing nude mice. The tumor inhibitory rates of cisplatin (10 mg/kg) co-treated with L-THP at 10, 20 and 40 mg/kg were 71.4%, 70.4% and 69.4%, respectively, in H460 tumor bearing nude mice, higher than that of in cisplatin alone group (60.6%), while in HCT116 tumor bearing nude mice, the tumor inhibitory rates in co-treated with 20 mg/kg L-THP was 34.7% (vs 26.3% in cisplatin alone group). Moreover, L-THP reduced cisplatin accumulation and alleviated cisplatin-induced cytotoxicity in human primary renal tubular cells. Therefore, our findings suggested that concomitant administration of L-THP could attenuate cisplatin-induced renal injury via selective inhibition of OCT2 without impairing its antitumor efficacy.
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http://dx.doi.org/10.1016/j.bcp.2020.114021DOI Listing
July 2020

CN128: A New Orally Active Hydroxypyridinone Iron Chelator.

J Med Chem 2020 04 2;63(8):4215-4226. Epub 2020 Apr 2.

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, Zhejiang Province, China.

Deferoxamine, deferiprone, and deferasirox are used for the treatment of systemic iron overload, although they possess limitations due to lack of oral activity, lower efficacy, and side effects. These limitations led to a search for an orally active iron chelator with an improved therapeutic index. The lower efficacy of deferiprone is due to rapid glucuronidation, leading to the formation of a nonchelating metabolite. Here, we demonstrate that the influence of metabolism can be reduced by introducing a sacrificial site for glucuronidation. A log -guided investigation of 20 hydroxpyridinones led to the identification of CN128. The Fe(III) affinity and metal selectivity of CN128 are similar to those of deferiprone, the log value is more lipophilic, and its iron scavenging ability is superior. Overall, CN128 was demonstrated to be safe in a range of toxicity assessments and is now in clinical trials for the treatment of β-thalassemia after regular blood transfusion.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00137DOI Listing
April 2020

Pregnancy Impacts Entecavir Pharmacokinetics but Does Not Alter Its Renal Excretion.

J Pharm Sci 2020 05 4;109(5):1811-1818. Epub 2020 Feb 4.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, P.R. China. Electronic address:

Entecavir (ETV) is a first-line antiviral drug against the hepatitis B virus. This study was designed to investigate whether ETV pharmacokinetics changes during pregnancy and the underlying mechanism. The results showed that ETV exposure in plasma was higher in pregnant rats than in nonpregnant rats, whereas the exposure after delivery was recovered to that in nonpregnant rats. Because 70% of orally dosed ETV is eliminated by kidney, the effects of estradiol (E2) and progesterone (P4), 2 important hormones during pregnancy, on ETV-related renal transporters were investigated. Our results revealed that the activities of the ETV-related renal transporters hOAT1, hOAT3, hMATE1, and hMATE2-K were clearly inhibited by E2 and P4, resulting in reduced ETV accumulation in transporter-transfected cell models. However, the cumulative urinary excretion of ETV in pregnant rats exhibited no significant difference compared to nonpregnant rats, although the endogenous creatinine clearance in pregnant rats was 1.5-fold that of nonpregnant rats. In conclusion, ETV plasma exposure is increased during pregnancy, but ETV renal excretion displays no significant alteration. This may be because, during pregnancy, increased glomerular ETV filtration compensated for the decrease in renal tubular ETV secretion that occurs by E2- and P4-mediated inhibition of related transporters.
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http://dx.doi.org/10.1016/j.xphs.2020.01.027DOI Listing
May 2020

Rapid quantification of tenofovir in umbilical cord plasma and amniotic fluid in hepatitis B mono-infected pregnant women during labor by ultra-performance liquid chromatography/tandem mass spectrometry.

Rapid Commun Mass Spectrom 2020 May;34(9):e8728

Key Laboratory of Clinical Cancer Pharmacology and Toxicology Research of Zhejiang Province, Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine, Zhejiang, Hangzhou, China.

Rationale: Tenofovir (TFV) is a first-line antiviral agent against hepatitis B virus (HBV) and is recommended for the prevention of mother-to-infant transmission of HBV. To study the distribution of TFV in umbilical cord plasma and amniotic fluid of HBV-infected pregnant women, a rapid and sensitive method for TFV determination was developed and validated.

Methods: The quantification method was developed using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). The analytes were separated on an Acquity UPLC HSS T3 column under gradient elution with methanol and 0.01% ammonia solution in 10 mM ammonium acetate/water. This is the first reported method for the determination of TFV using alkaline rather than acidic mobile phases. Linearity, accuracy, precision, limit of quantification, specificity and stability were assessed.

Results: Detection of TFV was achieved within 4 min. The calibration curves for TFV quantification showed excellent linearity in the range of 1-500 ng/mL. The intra- and interbatch precision and accuracy ranged from -4.35% to 6.92%. This method was successfully applied to determination of samples from 50 HBV mono-infected women undergoing tenofovir disoproxil fumarate therapy. The mean concentrations of TFV in the umbilical cord and amniotic fluid samples were 29.2 (4.6-86) and 470.9 (156-902) ng/mL, respectively, which showed a moderate positive correlation (r = 0.5299, P<0.001).

Conclusions: A simple, rapid but sensitive bioanalytical method to determine TFV concentration in both umbilical cord plasma and amniotic fluid using LC/MS/MS was developed and applied to HBV-infected women during labor who were undergoing TDF therapy, which will help us understand the efficacy and safety of tenofovir during pregnancy.
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http://dx.doi.org/10.1002/rcm.8728DOI Listing
May 2020

Dehydrocorydaline induced antidepressant-like effect in a chronic unpredictable mild stress mouse model via inhibiting uptake-2 monoamine transporters.

Eur J Pharmacol 2019 Dec 8;864:172725. Epub 2019 Oct 8.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Science, Zhejiang University, Hangzhou, 310058, China. Electronic address:

Dehydrocorydaline, is an active alkaloid compound in Corydalis yanhusuo W. T. Wang. We found dehydrocorydaline induced antidepressant-like effects in a chronic unpredictable mild stress mouse model, but the exact mechanisms have not been addressed. We speculated that dehydrocorydaline may have an antidepressant effect via inhibiting monoamine transporters in the brain. We evaluated the mechanism of action of dehydrocorydaline by examining the levels of monoamine transmitters (5-HT, NE and DA) in the prefrontal cortex in chronic unpredictable mild stress mice. Then, we used cell models and the mouse synaptosome to study molecular and cellular mechanisms underlying these behaviors and monoamine alterations by dehydrocorydaline. Our results indicated that dehydrocorydaline affects the concentrations of monoamine transmitters and decreases the turnover ratio, which indicates increased neuronal activity. The possible mechanism is that dehydrocorydaline potently inhibits uptake-2 transporters with the IC values of 0.1-4 μM and could inhibit the reuptake of 5-HT/DA/NE in the synaptosome. These data suggest that dehydrocorydaline has an antidepressant effect that is likely related to changing the content of monoamines in the brain by inhibiting uptake-2 transporters.
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http://dx.doi.org/10.1016/j.ejphar.2019.172725DOI Listing
December 2019

Up-regulation of hepatic fatty acid transporters and inhibition/down-regulation of hepatic OCTN2 contribute to olanzapine-induced liver steatosis.

Toxicol Lett 2019 Nov 19;316:183-193. Epub 2019 Aug 19.

Institute of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address:

Olanzapine, a representative of antipsychotics, is a first-line drug for treatment of schizophrenia. However, olanzapine-induced liver steatosis limits its clinical utilization. This study is to explore the mechanism of liver steatosis induced by olanzapine based on the regulation of transporters involved in uptake and oxidation of fatty acids. Our results revealed that 12-week oral administration of olanzapine increased hepatic triglyceride(TG), caused liver steatosis. Our further studies showed that the expression of fatty acid transporter 2(FATP2) and fatty acid binding protein 1(FABP1) were up-regulated in liver of female mice after 12-week olanzapine exposure, as well as in primary mouse hepatocytes treated with olanzapine. Olanzapine treatment also reduced hepatic β-hydroxybutyrate level (indicator of fatty acid β-oxidation), meanwhile, the L-carnitine (L-Car) concentration in liver of olanzapine group was significantly lower than that in control group. Further study demonstrated that both mRNA and protein expression of hepatic OCTN2 (carnitine/organic cation transporter 2) were obviously down-regulated in male mice after 12-week olanzapine treatment. Also, olanzapine markedly inhibited L-Car uptake in MDCK-hOCTN2 cells (1.06 μM of IC), HepG2 cells and primary mouse hepatocytes. Supplementation of L-Car attenuated hepatic TG rise and improved simple steatosis in olanzapine treatment mice. Taken together, up-regulation of FATP2/FABP1 and down-regulation/inhibition of hepatic OCTN2 probably contribute to olanzapine-induced liver steatosis. Supplementation of L-Car is a promising strategy to attenuate olanzapine-induced simple steatosis.
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http://dx.doi.org/10.1016/j.toxlet.2019.08.013DOI Listing
November 2019

Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells.

J Pharm Sci 2019 10 26;108(10):3416-3424. Epub 2019 Jun 26.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, P. R. China. Electronic address:

PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d-L-histidine, with K values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a K value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.
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http://dx.doi.org/10.1016/j.xphs.2019.06.016DOI Listing
October 2019

Roles of organic anion transporter 2 and equilibrative nucleoside transporter 1 in hepatic disposition and antiviral activity of entecavir during non-pregnancy and pregnancy.

Br J Pharmacol 2019 09 10;176(17):3236-3249. Epub 2019 Jul 10.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.

Background And Purpose: Entecavir (ETV), a first-line antiviral drug against hepatitis B virus (HBV), has the possibility to be used to prevent mother-to-child transmission. The aim of present study was to clarify the mechanism of ETV uptake into hepatocytes and evaluate the alteration of ETV's hepatic distribution during pregnancy.

Experimental Approach: The roles of equilibrative nucleotide transporter (ENT) 1 and organic anion transporter (OAT) 2 in ETV accumulation and anti-HBV efficacy were studied in human ENT1 or OAT2 overexpressed cell models and HepG2.2.15 cells, respectively; meanwhile, the liver-to-plasma ETV concentration ratios in non-pregnant and pregnant mice were measured to evaluate the effect of pregnancy on ETV hepatic distribution.

Key Results: ETV was shown to be a substrate of ENT1 and OAT2. An ENT1 inhibitor significantly decreased the efficacy of ETV in HepG2.2.15 cells, while overexpression of OAT2 increased susceptibility of HBV to ETV. The liver-to-plasma ETV concentration ratios in pregnant mice were sharply reduced; whereas, the absolute concentration of ETV in the liver did not obviously alter in pregnancy. Although oestradiol and progesterone showed a concentration-dependent inhibition on ETV accumulation both in hepatic cell lines and in primary human hepatocytes, a physiologically relevant concentration of oestradiol and progesterone did not affect antiviral activity of ETV.

Conclusions And Implications: OAT2 and ENT1 are the main transporters involved in the hepatic uptake and anti-HBV efficacy of ETV. The concentration of ETV in the liver was not obviously altered during pregnancy, which indicates that dosage adjustment in pregnancy is not necessary.
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http://dx.doi.org/10.1111/bph.14756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692639PMC
September 2019

Multiple Drug Transporters Contribute to the Placental Transfer of Emtricitabine.

Antimicrob Agents Chemother 2019 08 25;63(8). Epub 2019 Jul 25.

Women's Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

Emtricitabine (FTC) is a first-line antiviral drug recommended for the treatment of AIDS during pregnancy. We hypothesized that transporters located in the placenta contribute to FTC transfer across the blood-placenta barrier. BeWo cells, cell models with stable or transient expression of transporter genes, primary human trophoblast cells (PHTCs), and small interfering RNAs (siRNAs) were applied to demonstrate which transporters were involved. FTC accumulation in BeWo cells was reduced markedly by inhibitors of equilibrative nucleoside transporters (ENTs), concentrative nucleoside transporters (CNTs), organic cation transporters (OCTs), and organic cation/carnitine transporter 1 (OCTN1) and increased by inhibitors of breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs). ENT1, CNT1, OCTN1, MRP1/2/3, and BCRP, but not ENT2, CNT3, OCTN2, or multidrug resistance protein 1 (MDR1), were found to transport FTC. FTC accumulation in PHTCs was decreased significantly by inhibitors of ENTs and OCTN1. These results suggest that ENT1, CNT1, and OCTN1 probably contribute to FTC uptake from maternal circulation to trophoblasts and that ENT1, CNT1, and MRP1 are likely involved in FTC transport between trophoblasts and fetal blood, whereas BCRP and MRP1/2/3 facilitate FTC transport from trophoblasts to maternal circulation. Coexistence of tenofovir or efavirenz with FTC in the cell medium did not influence FTC accumulation in BeWo cells or PHTCs.
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http://dx.doi.org/10.1128/AAC.00199-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658773PMC
August 2019

Maternal Plasma l-Carnitine Reduction During Pregnancy Is Mainly Attributed to OCTN2-Mediated Placental Uptake and Does Not Result in Maternal Hepatic Fatty Acid -Oxidation Decline.

Drug Metab Dispos 2019 06 27;47(6):582-591. Epub 2019 Mar 27.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences (M.B., Y.C., M.C., P.L., Z.M., H.Z., S.Z., H.J.) and Women's Hospital, School of Medicine (Q.Z., D.S., C.Z.), Zhejiang University, Hangzhou, People's Republic of China

l-Carnitine (l-Car) plays a crucial role in fatty acid -oxidation. However, the plasma l-Car concentration in women markedly declines during pregnancy, but the underlying mechanism and its consequences on maternal hepatic -oxidation have not yet been clarified. Our results showed that the plasma l-Car level in mice at gestation day (GD) 18 was significantly lower than that in nonpregnant mice, and the mean fetal-to-maternal plasma l-Car ratio in GD 18 mice was 3.0. Carnitine/organic cation transporter 2 (OCTN2) was highly expressed in mouse and human placenta and upregulated as gestation proceeds in human placenta, whereas expressions of carnitine transporter (CT) 1, CT2, and amino acid transporter B were extremely low. Further study revealed that renal peroxisome proliferator-activated receptor (PPAR) and OCTN2 were downregulated and the renal l-Car level was reduced, whereas the urinary excretion of l-Car was lower in late pregnant mice than in nonpregnant mice. Meanwhile, progesterone (pregnancy-related hormone) downregulated the expression of renal OCTN2 via PPAR-mediated pathway, and inhibited the activity of OCTN2, but estradiol, corticosterone, and cortisol did not. Unexpectedly, the maternal hepatic level of l-Car and -hydroxybutyrate (an indicator of mitochondrial -oxidation), and mRNA levels of several enzymes involved in fatty acid -oxidation in GD 18 mice were higher than that in nonpregnant mice. In conclusion, OCTN2 mediated l-Car transfer across the placenta played a major role in maternal plasma l-Car reduction during pregnancy, which did not subsequently result in maternal hepatic fatty acid -oxidation decrease.
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http://dx.doi.org/10.1124/dmd.119.086439DOI Listing
June 2019

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

Xenobiotica 2019 Oct 9;49(10):1237-1243. Epub 2019 Jan 9.

a Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences , Zhejiang University , Hangzhou , PR China.

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. uptake tests showed that jatrorrhizine strongly inhibited PMAT-mediated MPP uptake with an IC value of 1.05 μM and reduced 5-HT and NE uptake mediated by hOCT2, hOCT3 and hPMAT with IC 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.
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http://dx.doi.org/10.1080/00498254.2018.1542188DOI Listing
October 2019

Clozapine-induced reduction of l-carnitine reabsorption via inhibition/down-regulation of renal carnitine/organic cation transporter 2 contributes to liver lipid metabolic disorder in mice.

Toxicol Appl Pharmacol 2019 01 19;363:47-56. Epub 2018 Nov 19.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China. Electronic address:

Clozapine, an atypical antipsychotic drug, is widely utilized for the treatment of schizophrenia; however, clozapine-induced metabolic disorders, such as fatty liver and weight gain, warrant increased attention. Considering the crucial role of l-carnitine (L-Car) in fatty acid oxidation and carnitine/organic cation transporter (OCTN) 2 in renal reabsorption of L-Car, we aimed to study whether clozapine-induced liver lipid metabolic disorder is associated with L-Car dysregulation via inhibition/down-regulation of renal OCTN2. Our results reveal that clozapine inhibits L-Car uptake in MDCK-hOCTN2 cells with an IC value of 1.78 μM. Additionally, clozapine significantly reduces the uptake of L-Car in HK-2 cells, mouse primary cultured proximal tubular (mPCPT) cells and HepG2 cells. Acute (intraperitoneal injection) and 21-day successive oral administration of clozapine at 12.5, 25, and 50 mg/kg to mice resulted in 2-3-fold greater renal excretion of L-Car than in the vehicle group, and the concentration of L-Car in plasma and liver was significantly decreased. Concomitantly, mRNA and protein levels of mOctn2 in the kidney were markedly down regulated. Additionally, 28-day oral administration of clozapine induced increased triglyceride (TG) and total cholesterol (TCHO) levels in mouse livers, while L-Car (40 mg/kg - 1 g/kg) attenuated clozapine-induced liver TG and TCHO increase in a dose-dependent manner. These results indicate that clozapine-induced reduction of L-Car reabsorption via inhibition/down-regulation of renal OCTN2 contributes to liver lipid metabolic disorder. L-Car supplementation is probably an effective strategy to attenuate clozapine-induced abnormal lipid metabolism.
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http://dx.doi.org/10.1016/j.taap.2018.11.007DOI Listing
January 2019

Co-administration of nuciferine reduces the concentration of metformin in liver via differential inhibition of hepatic drug transporter OCT1 and MATE1.

Biopharm Drug Dispos 2018 Nov 30;39(9):411-419. Epub 2018 Oct 30.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

Nuciferine (NF), one of the main and effective components in Nelumbo nucifera Gaertn. leaf extracts, is a promising drug candidate for the treatment of obesity-related diseases, while metformin is a first line therapeutic drug for type 2 diabetes mellitus. Since nuciferine and metformin are likely to be co-administered, the aim of the present study was to evaluate whether co-administration of nuciferine would influence the liver (target tissue) distribution and the anti-diabetic effect of metformin by inhibiting hepatic organic cation transporter 1 (OCT1) and multidrug and toxin extrusion 1 (MATE1). The data demonstrated that nuciferine significantly reduced metformin accumulation in MDCK cells stably expressing human OCT1 (MDCK-hOCT1) or hMATE1 (MDCK-hMATE1), and primary cultured mouse hepatocytes. Furthermore, the presence of nuciferine in the basal compartment caused a concentration-dependent reduction of intracellular metformin accumulation in MDCK-hOCT1/hMATE1 cell monolayers. Compared with the metformin treatment-alone group, co-administration of nuciferine (40 mg/kg) markedly reduced the metformin concentration in mouse livers at 30 and 60 min after a single oral dose of metformin (200 mg/kg), and subsequently impaired the glucose-lowering effect of metformin (200 mg/kg), but the glucose-lowering effect became no different at 90 and 120 min. Therefore, nuciferine influenced the liver concentration and glucose-lowering effect of metformin only for a period of time after dose, administration of nuciferine and metformin with an interval might prevent the drug-drug interaction mediated by OCT1 and MATE1.
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http://dx.doi.org/10.1002/bdd.2158DOI Listing
November 2018

Regulation and biological role of the peptide/histidine transporter SLC15A3 in Toll-like receptor-mediated inflammatory responses in macrophage.

Cell Death Dis 2018 07 10;9(7):770. Epub 2018 Jul 10.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang, China.

The peptide/histidine transporter SLC15A3 is responsible for transporting histidine, certain dipeptide and peptidomimetics from inside the lysosome to cytosol. Previous studies have indicated that SLC15A3 transcripts are mainly expressed in the lymphatic system, however, its regulation and biological role in innate immune responses and inflammatory diseases are as yet unknown. In this study, mouse peritoneal macrophages (PMs), mouse bone marrow-derived macrophages (BMDMs), the human acute monocytic leukemia cell line THP-1 and the human lung epithelial carcinoma cell line A549 were used to investigate the regulation and biological role of SLC15A3 in TLR-mediated inflammatory responses. Our results showed that SLC15A3 was upregulated by TLR2, TLR4, TLR7 and TLR9 ligands in macrophages at both the mRNA and protein levels via activation of NF-κB (nuclear factor-kappa-B), MAPK (mitogen-activated protein kinase) and IRF3 (interferon regulatory factor 3). Furthermore, knockdown or overexpression of SLC15A3 influenced the TLR4-triggered expression of proinflammatory cytokines. A reporter gene assay showed that the SLC15A3 promotor contained potential NF-κB binding sites, which were reasonable for regulating SLC15A3 by TLR-activation through NF-κB signaling. Additionally, SLC15A3 expression was increased and positively related to inflammation in mice with bacterial peritonitis. The collective findings suggest that SLC15A3 is regulated by various TLRs, and that it plays an important role in regulating TLR4-mediated inflammatory responses.
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http://dx.doi.org/10.1038/s41419-018-0809-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039463PMC
July 2018

SLC15A2 and SLC15A4 Mediate the Transport of Bacterially Derived Di/Tripeptides To Enhance the Nucleotide-Binding Oligomerization Domain-Dependent Immune Response in Mouse Bone Marrow-Derived Macrophages.

J Immunol 2018 07 21;201(2):652-662. Epub 2018 May 21.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109;

There is increasing evidence that proton-coupled oligopeptide transporters (POTs) can transport bacterially derived chemotactic peptides and therefore reside at the critical interface of innate immune responses and regulation. However, there is substantial contention regarding how these bacterial peptides access the cytosol to exert their effects and which POTs are involved in facilitating this process. Thus, the current study proposed to determine the (sub)cellular expression and functional activity of POTs in macrophages derived from mouse bone marrow and to evaluate the effect of specific POT deletion on the production of inflammatory cytokines in wild-type, knockout and knockout mice. We found that PEPT2 and PHT1 were highly expressed and functionally active in mouse macrophages, but PEPT1 was absent. The fluorescent imaging of muramyl dipeptide-rhodamine clearly demonstrated that PEPT2 was expressed on the plasma membrane of macrophages, whereas PHT1 was expressed on endosomal membranes. Moreover, both transporters could significantly influence the effect of bacterially derived peptide ligands on cytokine stimulation, as shown by the reduced responses in knockout and knockout mice as compared with wild-type animals. Taken as a whole, our results point to PEPT2 (at plasma membranes) and PHT1 (at endosomal membranes) working in concert to optimize the uptake of bacterial ligands into the cytosol of macrophages, thereby enhancing the production of proinflammatory cytokines. This new paradigm offers significant insight into potential drug development strategies along with transporter-targeted therapies for endocrine, inflammatory, and autoimmune diseases.
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http://dx.doi.org/10.4049/jimmunol.1800210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039277PMC
July 2018

Development and Validation of a HPLC-ESI-MS/MS Method for Simultaneous Quantification of Fourteen Alkaloids in Mouse Plasma after Oral Administration of the Extract of Corydalis yanhusuo Tuber: Application to Pharmacokinetic Study.

Molecules 2018 Mar 21;23(4). Epub 2018 Mar 21.

College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

The tuber of is a famous traditional Chinese medicine and found to have potent pharmacological effects, such as antinociceptive, antitumor, antibacterial, anti-inflammatory, and anti-depressive activities. Although there are several methods to be developed for the analysis and detection of the bioactive ingredients' alkaloids, so far, only few prominent alkaloids could be quantified, and in vitro and in vivo changes of comprehensive alkaloids after oral administration are still little known. In this study, we first developed a simple and sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) method to quantify the comprehensive alkaloids of extracts of in mouse plasma, using nitidine chloride as an internal standard. As results, at least fourteen alkaloids, including an aporphine (oxoglaucine), a protopine (protopine), five tertiary alkaloids (corydaline, tetrahydroberberine, tetrahydropalmatine, tetrahydrocolumbamine, and tetrahydrocoptisine) and seven quaternary alkaloids (columbamine, palmatine, berberine, epiberberine, coptisine, jatrorrhizine, and dehydrocorydaline) could be well quantified simultaneously in mouse plasma. The lower limits of quantification were greater than, or equal to, 0.67 ng/mL, and the average matrix effects ranged from 96.4% to 114.3%. The mean extraction recoveries of quality control samples were over 71.40%, and the precision and accuracy were within the acceptable limits. All the analytes were shown to be stable under different storage conditions. Then the established method was successfully applied to investigate the pharmacokinetics of these alkaloids after oral administration of the extract of in mice. To the best of our knowledge, this is the first document to report the comprehensive and simultaneous analyses of alkaloids of in mouse plasma. It was efficient and useful for comprehensive pharmacokinetic and metabolomic analyses of these complex alkaloids after drug administration.
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http://dx.doi.org/10.3390/molecules23040714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017933PMC
March 2018

Expression and regulation of proton-coupled oligopeptide transporters in colonic tissue and immune cells of mice.

Biochem Pharmacol 2018 02 3;148:163-173. Epub 2018 Jan 3.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA. Electronic address:

A number of studies have implicated proton-coupled oligopeptide transporters (POTs) in the initiation and/or progression of inflammatory bowel disease and immune cell signaling. With this in mind, the aim of this study was to delineate the expression of POTs in mouse colonic tissues and immune cells, and characterize the potential role of these transporters in nucleotide-binding oligomerization domain (NOD) signaling. Using a dextran sodium sulfate (DSS)-induced colitis mouse model, we found that DSS down regulated Pht1 gene expression and up regulated Pht2 gene expression in colonic tissue and immune cells. In contrast, PEPT1 protein was absent from the colonic tissue and immune cells of normal and DSS-treated mice. NOD ligands, muramyl dipeptide (MDP) and l-Ala-γ-d-Glu-meso-diaminopimelic acid (tri-DAP), were shown to be substrates of PHT2 in MDCK-hPHT2 cells. Subsequent studies revealed that the immune response of lamina propia mononuclear cells may be regulated by PHT1 and PHT2, and that PHT2 facilitated the NOD-dependent immune response in RAW264.7 macrophages. These results clarified the expression of POTs in mouse colonic segments, cells and subtypes, and the role of increased Pht2 expression during chemically-induced colitis in facilitating NOD-dependent immune response. The findings further suggest that intestinal PHT2 may serve as a therapeutic target for IBD therapy.
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http://dx.doi.org/10.1016/j.bcp.2017.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801143PMC
February 2018

Functional Characterization of Human Peptide/Histidine Transporter 1 in Stably Transfected MDCK Cells.

Mol Pharm 2018 02 2;15(2):385-393. Epub 2018 Jan 2.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University , Zhejiang 310058, China.

The proton-coupled oligopeptide transporter PHT1 (SLC15A4), which facilitates cross-membrane transport of histidine and small peptides from inside the endosomes or lysosomes to cytosol, plays an important role in intracellular peptides homeostasis and innate immune responses. However, it remains a challenge to elucidate functional properties of the PHT1 transporter because of its subcellular localization. The purpose of this study was to resort hPHT1 protein from the subcellular to outer cell membrane of MDCK cells stably transfected with human PHT1 mutants, and to characterize its functional activity in these cells. Using this model, the functional activity of hPHT1 was evaluated by cellular uptake studies with d-l-histidine, GlySar, and the bacterial peptidoglycan products MDP and Tri-DAP. We found that the disruption of two dileucine motifs was indispensable for hPHT1 transporter being preferentially targeting to plasma membranes. hPHT1 showed high affinity for d-l-histidine and low affinity for GlySar, with K values of 16.3 ± 1.9 μM and 1.60 ± 0.30 mM, respectively. Moreover, the bacterial peptidoglycan components MDP and Tri-DAP were shown conclusively to be hPHT1 substrates. The uptake of MDP by hPHT1 was inhibited by di/tripeptides and peptide-like drugs, but not by glycine and acyclovir. The functional activity of hPHT1 was also pH-dependent, with an optimal cellular uptake in buffer pH 6.5. Taken together, we established a novel cell model to evaluate the function of hPHT1 in vitro, and confirmed that MDP and Tri-DAP were substrates of hPHT1. Our findings suggest that PHT1 may serve as a potential target for reducing the immune responses and for drug treatment of inflammatory diseases.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826766PMC
February 2018

Multiple organic cation transporters contribute to the renal transport of sulpiride.

Biopharm Drug Dispos 2017 Dec 20;38(9):526-534. Epub 2017 Nov 20.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

Sulpiride, a selective dopamine D2 receptor blocker, is used widely for the treatment of schizophrenia, depression and gastric/duodenal ulcers. Because the great majority of sulpiride is positively charged at physiological pH 7.4, and ~70% of the dose recovered in urine is in the unchanged form after human intravenous administration of sulpiride, it is believed that transporters play an important role in the renal excretion of sulpiride. The aim of the present study was to explore which transporters contribute to the renal disposition of sulpiride. The results demonstrated that sulpiride was a substrate of human carnitine/organic cation transporter 1 (hOCTN1) and 2 (hOCTN2), human organic cation transporter 2 (hOCT2), human multidrug and toxin efflux extrusion protein 1 (hMATE1) and 2-K (hMATE2-K). Sulpiride accumulation from the basolateral (BL) to the apical (AP) side in MDCK-hOCT2/pcDNA3.1 cell monolayers was much greater than that in MDCK-hOCT2/hMATE1 cells, and cimetidine dramatically reduced the intracellular accumulation of sulpiride from BL to AP. In addition, the accumulation of sulpiride in mouse primary renal tubular cells (mPRTCs) was markedly reduced by inhibitors of Oct2 and Octns. The results implied that OCTN1, OCTN2, OCT2, MATE1 and MATE2-K probably contributed to the renal transfer of sulpiride, in which OCT2 mediated the uptake of sulpiride from the bloodstream to the proximal tubular cells, while MATEs contributed to the sulpiride efflux from the proximal tubular cells to the renal lumen, and OCTNs participated in both renal secretion and reabsorption.
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http://dx.doi.org/10.1002/bdd.2104DOI Listing
December 2017

Multiple drug transporters mediate the placental transport of sulpiride.

Arch Toxicol 2017 Dec 9;91(12):3873-3884. Epub 2017 Jun 9.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China.

Sulpiride is a typical antipsychotic drug for the treatment of schizophrenia, depression and other psychological disorders. It has been proven that a small amount of sulpiride could cross the human placenta using an ex vivo placental perfusion model. However, the placental transfer mechanism has not been elucidated. Considering the structure of sulpiride, we speculated that the transporters expressed in placenta might be involved in sulpiride uptake across the blood-placenta barrier. The aim of our study was to determine which transporters contributed to the placental transfer of sulpiride. Our results revealed that sulpiride was a substrate of human organic cation transporter (hOCT) 3, human multidrug resistance protein (hMDR) 1 and human breast cancer resistance protein (hBCRP) using transfected cells expressing respective transporters. In addition, the accumulation of sulpiride in BeWo cells (a human choriocarcinoma cell line) was obviously affected by inhibitors of carnitine/organic cation transporter (OCTN) 2, MDR1 and BCRP. The accumulation of sulpiride in primary human trophoblast cells was obviously affected by inhibitors of OCT3, OCTN1 and OCTN2. The above results indicate that hOCTN1 and hOCTN2 likely contribute to the sulpiride uptake from maternal circulation to trophoblast cells, while hMDR1 and hBCRP mediate the efflux from trophoblast cells to maternal circulation, and hOCT3 probably is involved in the bidirectional transport of sulpiride between the placenta and fetal blood.
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http://dx.doi.org/10.1007/s00204-017-2008-8DOI Listing
December 2017

Response to Comment on "Epigenetic activation of the drug transporter OCT2 sensitizes renal cell carcinoma to oxaliplatin".

Sci Transl Med 2017 05;9(391)

Institute of Drug and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

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http://dx.doi.org/10.1126/scitranslmed.aam6298DOI Listing
May 2017

Multiple SLC and ABC Transporters Contribute to the Placental Transfer of Entecavir.

Drug Metab Dispos 2017 03 6;45(3):269-278. Epub 2017 Jan 6.

Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (Z.M., X.Y., T.J., M.B., S.Z., H.J.); and Women's Hospital School of Medicine Zhejiang University, Hangzhou, China (C.Z., D.S.)

Entecavir (ETV), a nucleoside analog with high efficacy against hepatitis B virus, is recommended as a first-line antiviral drug for the treatment of chronic hepatitis B. However, scant information is available on the use of ETV in pregnancy. To better understand the safety of ETV in pregnant women, we aimed to demonstrate whether ETV could permeate placental barrier and the underlying mechanism. Our study showed that small amount of ETV could permeate across placenta in mice. ETV accumulation in activated or nonactivated BeWo cells (treated with or without forskolin) was sharply reduced in the presence of 100 µM of adenosine, cytidine, and in Na free medium, indicating that nucleoside transporters possibly mediate the uptake of ETV. Furthermore, ETV was proved to be a substrate of concentrative nucleoside transporter (CNT) 2 and CNT3, of organic cation transporter (OCT) 3, and of breast cancer resistance protein (BCRP) using transfected cells expressing respective transporters. The inhibition of ETV uptake in primary human trophoblast cells further confirmed that equilibrative nucleoside transporter (ENT) 1/2, CNT2/3, OCT3, and organic cation/carnitine transporter (OCTN) 2 might be involved in ETV transfer in human placenta. Therefore, ETV uptake from maternal circulation to trophoblast cells was possibly transported by CNT2/3, ENT1/2, and OCTN2, whereas ETV efflux from trophoblast cells to fetal circulation was mediated by OCT3, and efflux from trophoblast cells to maternal circulation might be mediated by BCRP, multidrug resistance-associated protein 2, and P-glycoprotein. The information obtained in the present study may provide a basis for the use of ETV in pregnancy.
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http://dx.doi.org/10.1124/dmd.116.073304DOI Listing
March 2017

Species Differences in Human and Rodent PEPT2-Mediated Transport of Glycylsarcosine and Cefadroxil in Pichia Pastoris Transformants.

Drug Metab Dispos 2017 02 11;45(2):130-136. Epub 2016 Nov 11.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, Michigan (Y.H., D.E.S.); and Laboratory of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China (F.S., H.J.)

The proton-coupled oligopeptide transporter PEPT2 (SLC15A2) plays an important role in the disposition of di/tripeptides and peptide-like drugs in kidney and brain. However, unlike PEPT1 (SLC15A1), there is little information about species differences in the transport of PEPT2-mediated substrates. The purpose of this study was to determine whether PEPT2 exhibited a species-dependent uptake of glycylsarcosine (GlySar) and cefadroxil using yeast Pichia pastoris cells expressing cDNA from human, mouse, and rat. In such a system, the functional activity of PEPT2 was evaluated with [H]GlySar as a function of time, pH, substrate concentration, and specificity, and with [H]cefadroxil as a function of concentration. We observed that the uptake of GlySar was pH-dependent with an optimal uptake at pH 6.5 for all three species. Moreover, GlySar showed saturable uptake kinetics, with K values in human (150.6 µM) > mouse (42.8 µM) ≈ rat (36.0 µM). The PEPT2-mediated uptake of GlySar in yeast transformants was specific, being inhibited by di/tripeptides and peptide-like drugs, but not by amino acids and nonsubstrate compounds. Cefadroxil also showed a saturable uptake profile in all three species, with K values in human (150.8 μM) > mouse (15.6 μM) ≈ rat (11.9 μM). These findings demonstrated that the PEPT2-mediated uptake of GlySar and cefadroxil was specific, species dependent, and saturable. Furthermore, based on the K values, mice appeared similar to rats but both were less than optimal as animal models in evaluating the renal reabsorption and pharmacokinetics of peptides and peptide-like drugs in humans.
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http://dx.doi.org/10.1124/dmd.116.073320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5267517PMC
February 2017
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