Publications by authors named "Feifeng Song"

17 Publications

  • Page 1 of 1

The role of alcohol dehydrogenase 1C in regulating inflammatory responses in ulcerative colitis.

Biochem Pharmacol 2021 Jul 19;192:114691. Epub 2021 Jul 19.

Clinical Pharmacy Center, Department of Pharmacy, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou 310014, Zhejiang, China; Key Laboratory of Endocrine Gland Diseases of Zhejiang Province, Hangzhou 310014, Zhejiang, China. Electronic address:

Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease caused by an interaction of genetics, immune responses, and environmental factors. However, the precise pathogenesis of UC remains poorly understood. The aim of the present study was to explore the potential target genes implicated in UC and to elucidate its underlying pathogenic mechanism. Firstly, three UC-associated microarray datasets (GSE75214, GSE87466, and GSE92517) were obtained to screen the differentially expressed genes (DEGs). As a result, alcohol dehydrogenase 1C (ADH1C) was the most significantly downregulated DEG in UC. We confirmed that ADH1C was downregulated in colonic tissue taken from patients with UC and colitis mice. Moreover, ADH1C expression was also decreased in colonic cell lines (NCM460 and HT29) treated with mixed proinflammatory cytokines (TNF-α, IFN-γ, IL-1β). Interestingly, we found that overexpression of ADH1C could distinctly decrease the production of IL-6 and IL-8. To elucidate the molecular mechanism of ADH1C in UC, gene set enrichment analysis (GSEA) was performed and demonstrated that immune-related pathways were mainly enriched in the low ADH1C group. Further studies displayed that STAT1/NF-κB pathway was activated in colitis mice and inflammatory cell model. Importantly, overexpression of ADH1C could suppress the phosphorylation of STAT1 and IκB. Therefore, ADH1C might regulate inflammatory responses through STAT1/NF-κB pathway. In conclusion, ADH1C was downregulated during inflammation, and its increased expression could inhibit the activation of STAT1/NF-κB pathway, thereby alleviating the secretion of IL-6 and IL-8. These findings indicate that ADH1C may be a potential therapeutic target for UC therapy.
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http://dx.doi.org/10.1016/j.bcp.2021.114691DOI Listing
July 2021

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 07 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

Low curcumin concentration enhances the anticancer effect of 5-fluorouracil against colorectal cancer.

Phytomedicine 2021 May 17;85:153547. Epub 2021 Mar 17.

Department of Pharmacy, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China. Electronic address:

Background: Colon cancer treatments include surgery, radiotherapy, and chemotherapy. Chemotherapy using 5-fluorouracil (5-FU) has been widely applied to treat colorectal cancer (CRC). However, it is important to explore the use of chemotherapy drugs in combination with other agents to decrease severe adverse effects.

Purpose: This study aimed to investigate the effects of curcumin in combination with 5-FU on the proliferation, migration, and apoptosis of CRC SW620 cell line both in vitro and in vivo.

Methods: Flow cytometry was used to study the effect of curcumin on chemotherapy-induced apoptosis in CRC cells. The mechanism of curcumin's enhanced antitumor effect in vivo was investigated using gene knockdown, TUNEL, western blot, qRT-PCR and immunohistochemistry.

Results: The results showed a synergistic effect of the two compounds on CRC cells. Considerable reduction in the proliferation and migration of SW620 cells was observed in the combination treatment group. Significantly increased apoptosis rate extended the survival of immunodeficient mice in the combination group as compared to that of the 5-FU group (p < 0.05). The results showed that curcumin significantly inhibited pERK signaling and downregulated L1 expression in SW620 cells.

Conclusions: We conclude that curcumin promotes chemosensitivity of CRC cells to 5-FU by downregulating L1 expression. Our findings provide experimental evidence for the synergism between curcumin and 5-FU, which can be utilized in clinical applications for reducing the toxicity and adverse effects of 5-FU.
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http://dx.doi.org/10.1016/j.phymed.2021.153547DOI Listing
May 2021

Case report: severe myoclonus associated with oral midodrine treatment for hypotension.

Medicine (Baltimore) 2020 Oct;99(40):e21533

Department of Nephrology, Zhejiang Provincial People's Hospital.

Rationale: Midodrine is widely used in the treatment of hypotensive states, there have been no reports of myoclonus associated with midodrine use in hypotension with chronic kidney disease.

Patient Concerns: We report a 58-year-old female patient with chronic kidney disease (CKD) presenting with involuntary tremor 2 h after taking midodrine, which became more frequent after 6 h. Brain CT and neurological examination did not yield findings of note. Blood chemistry showed serum albumin of 3.1 g/L, ALT of 19 U/L, AST of 22 U/L, SCr of 273.9 μmol/L, K of 2.94 mmol/L, Ca of 1.63 mmol/L, and Mg of 0.46 mmol/L. Her BP was maintained at 83-110/56-75 mmHg. Her urine volume was 600-1000 mL/d, and her heart rate was within a range of 90-100 beats/min.

Diagnosis: Chronic kidney disease (CKD), hypotension, metabolic acidosis, hypocalcemia, hypokalemia, and hypomagnesemia.

Interventions: Midodrine treatment was stopped and the patient was treated with intravascular rehydration and furosemide. Myoclonus ceased one day after midodrine withdrawal.

Lessons: Oral midodrine is widely used in the treatment of orthostatic hypotension, recurrent reflex syncope and dialysis-associated hypotension and the adverse effects are mostly mild. However, clinicians should be alert for midodrine-induced myoclonus, especially in patients with CKD.
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http://dx.doi.org/10.1097/MD.0000000000021533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7535667PMC
October 2020

High-Throughput Sequencing-Based Analysis of T Cell Repertoire in Lupus Nephritis.

Front Immunol 2020 6;11:1618. Epub 2020 Aug 6.

Lin He's Academician Workstation of New Medicine and Clinical Translation at The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.

T cell receptor (TCR)-mediated immune functions are closely related to autoimmune diseases, such as systemic lupus erythematosus (SLE). However, technical challenges used to limit the accurate profiling of TCR diversity in SLE and the characteristics of SLE patients remain largely unknown. In this study, we collected peripheral blood samples from 10 SLE patients with lupus nephritis (LN) who were confirmed by renal biopsy, as well as 10 healthy controls. The TCR repertoire of each sample was assessed by high-throughput sequencing to examine the distinction between SLE subjects and healthy controls. Our results showed statistically significant differences in TCR diversity and usage of TRBV/TRBJ genes between the two groups. A set of signature V-J combinations enabled efficient identification of SLE cases, yielding an area under the curve (AUC) of 0.89 (95% CI: 0.74-1.00). Taken together, our results revealed the potential correlation between the TCR repertoire and SLE status, which may facilitate the development of novel immune biomarkers.
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http://dx.doi.org/10.3389/fimmu.2020.01618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423971PMC
April 2021

Identification of key microRNAs and hub genes in non-small-cell lung cancer using integrative bioinformatics and functional analyses.

J Cell Biochem 2020 03 6;121(3):2690-2703. Epub 2019 Nov 6.

Department of Pharmacy, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, China.

Non-small-cell lung cancer (NSCLC) is an extremely debilitating respiratory malignancy. However, the pathogenesis of NSCLC has not been fully clarified. The main objective of our study was to identify potential microRNAs (miRNAs) and their regulatory mechanism in NSCLC. Using a systematic review, two NSCLC-associated miRNA data sets (GSE102286 and GSE56036) were obtained from Gene Expression Omnibus, and the differentially expressed miRNAs (DE-miRNAs) were accessed by GEO2R. Survival analysis of candidate DE-miRNAs was conducted using the Kaplan-Meier plotter database. To further illustrate the roles of DE-miRNAs in NSCLC, their potential target genes were predicted by miRNet and were annotated by the Database for Annotation, Visualization and Integrated Discovery (DAVID) program. Moreover, the protein-protein interaction (PPI) and miRNA-hub gene regulatory network were established using the STRING database and Cytoscape software. The function of DE-miRNAs in NSCLC cells was evaluated by transwell assay. Compared with normal tissues, a total of eight DE-miRNAs was commonly changed in two data sets. The survival analysis showed that six miRNAs (miR-21-5p, miR-31-5p, miR-708-5p, miR-30a-5p, miR-451a, and miR-126-3p) were significantly correlated with overall survival. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that target genes of upregulated miRNAs were enriched in pathways in cancer, microRNAs in cancer and proteoglycans in cancer, while the target genes of downregulated miRNAs were mainly associated with pathways in cancer, the PI3K-Akt signaling pathway and HTLV-I infection. Based on the miRNA-hub gene network and expression analysis, PTEN, EGFR, STAT3, RHOA, VEGFA, TP53, CTNNB1, and KRAS were identified as potential target genes. Furthermore, all six miRNAs exhibited significant effects on NSCLC cell invasion. These findings indicate that six DE-miRNAs and their target genes may play important roles in the pathogenesis of NSCLC, which will provide novel information for NSCLC treatments.
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http://dx.doi.org/10.1002/jcb.29489DOI Listing
March 2020

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

Effect of biphenyl hydrolase-like (BPHL) gene disruption on the intestinal stability, permeability and absorption of valacyclovir in wildtype and Bphl knockout mice.

Biochem Pharmacol 2018 10 17;156:147-156. Epub 2018 Aug 17.

Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Biphenyl hydrolase-like protein (BPHL) is a novel human serine hydrolase that was originally cloned from a breast carcinoma cDNA library and shown to convert valacyclovir to acyclovir and valganciclovir to ganciclovir. However, the exclusivity of this process has not been determined and, indeed, it is possible that a number of esterases/proteases may mediate the hydrolysis of valacyclovir and similar prodrugs. The objectives of the present study were to evaluate the in situ intestinal permeability and stability of valacyclovir in wildtype (WT) and Bphl knockout (KO) mice, as well as the in vivo oral absorption and intravenous disposition of valacyclovir and acyclovir in the two mouse genotypes. We found that Bphl knockout mice had no obvious phenotype and that Bphl ablation did not alter the jejunal permeability of valacyclovir during in situ perfusions (i.e., 0.54 × 10 in WT vs. 0.53 × 10 cm/s in KO). Whereas no meaningful changes occurred between genotypes in the gene expression of proton-coupled oligopeptide transporters (i.e., PepT1, PepT2, PhT1, PhT2), enzymatic upregulation of Cyp3a11, Cyp3a16, Abhd14a and Abhd14b was observed in some tissues of Bphl knockout mice. Most importantly, we found that valacyclovir was rapidly and efficiently hydrolyzed to acyclovir in the absence of BPHL, and that hydrolysis was more extensive after the oral vs. intravenous route of administration (for both genotypes). Taken as a whole, BPHL is not obligatory for the conversion of valacyclovir to acyclovir either presystemically or systemically.
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http://dx.doi.org/10.1016/j.bcp.2018.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188833PMC
October 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

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

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

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

Xenobiotica 2016 2;46(2):175-83. Epub 2015 Jul 2.

a 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.

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 Km of 0.273-5.80 μM, whereas berberrubine was a substrate for hOCT1 and hOCT2, but not for hOCT3, the Km 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 Clint (Vmax/Km) 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.
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http://dx.doi.org/10.3109/00498254.2015.1056283DOI Listing
November 2016

Simple and rapid determination of unsaturated fatty acids in 1 µl of rat plasma by LC-MS/MS.

Bioanalysis 2015 ;7(9):1081-91

Background: Deficiency or imbalance of unsaturated fatty acids will promote the pathogenesis of many diseases. In order to monitor the exposure of unsaturated fatty acids, the method based on LC-MS/MS was developed.

Results: Standard calibration curves for α-linolenic acid, linoleic acid, palmitoleic acid and oleic acid were linear (r ≥0.99). The intra-and interbatch accuracy (RE%) ranged from -4.5 to 8.6%, while the intra- and interbatch precisions (RSD%) were ≤8.7%. The extraction recovery varied from 85.4 to 99.6%, and no obvious matrix effect was observed.

Conclusion: The method offers a simple approach for measuring 4 unsaturated fatty acids in 1 μl rat plasma within 3.95 min.
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http://dx.doi.org/10.4155/bio.15.44DOI Listing
February 2016

Expression and regulation of the proton-coupled oligopeptide transporter PhT2 by LPS in macrophages and mouse spleen.

Mol Pharm 2014 Jun 2;11(6):1880-8. Epub 2014 May 2.

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.

Membrane transporter PhT2 (SLC15A3), which belongs to the proton-coupled oligopeptide transporter family, mediates the transport of di/tripeptides and histidine utilizing an inwardly directed proton gradient and negative membrane potential. The aim of this study was to elucidate the molecular expression of PhT2 in macrophages and mouse tissues and to explore the regulation of PhT2 by lipopolysaccharide (LPS). The results showed relatively high expression of PhT2 in J774A.1 and THP-1 macrophage cells, mouse spleen, and lung. Using an LPS-induced inflammatory cell model, we found that hPhT2 mRNA expression was up-regulated in THP-1 cells and that the up-regulation was suppressed by pyrrolidine dithiocarbamate, a specific inhibitor of NF-κB. Similar results were observed in mouse spleen during LPS-induced acute inflammation. Using dual-labeling immunofluorescence and confocal laser scanning microscopy, we confirmed that mPhT2 was colocalizing with lysosome-associated membrane protein 1 in transfected HEK293 cells. These results suggested that PhT2, a lysosomal membrane transporter, was up-regulated by LPS via the NF-κB signaling pathway.
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http://dx.doi.org/10.1021/mp500014rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051248PMC
June 2014

In vitro interaction of clopidogrel and its hydrolysate with OCT1, OCT2 and OAT1.

Int J Pharm 2014 Apr 11;465(1-2):5-10. Epub 2014 Feb 11.

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:

Clopidogrel (CP) is metabolized by CYPs to the active metabolite, or hydrolyzed by esterase to clopidogrel carboxylate (CPC) in liver, and CPC is partly excreted from urine. Therefore, the objective of the present study was to evaluate the interactions of CP and CPC with organic cation transporter 1 (OCT1) (in liver), and CPC with organic cation transporter 2 (OCT2) and organic anion transporter 1 (OAT1) (in kidney). Both CP and CPC inhibited the uptake of 1-methyl-4-phenylpyridinium (MPP(+)) and metformin, typical substrates of OCT1, in MDCK-hOCT1 cells with low IC₅₀ (0.307-14.0 μM). CPC (100 μM) reduced the uptake of MPP(+) and metformin mediated by OCT2 in MDCK-hOCT2 cells to 60.8% and 33.6% of the control, CPC (500 μM) decreased the uptake of 6-carboxyfluorescein (6-CFL) and para-aminohippuric acid (PAH), substrates of OAT1, in MDCK-hOAT1 cells to 64.6% and 79.4% of the control. CP and CPC were also found to inhibit other drugs of OCT1 substrates, such as lamivudine and amantadine, in MDCK-hOCT1 cells with the IC₅₀ of 1.97-4.15μM, except CPC on amantadine (IC₅₀>100 μM). The inhibition of CP and CPC on lamivudine uptake in primary rat hepatocytes was also confirmed with the IC₅₀ of 2.91 and 1.25μM, respectively. Additionally, CP and CPC were not substrates of OCT1 and OCT2, whereas CPC was a substrate of OAT1 with the Km of 5.61 μM. In conclusion, CP and CPC are strong inhibitors of OCT1, but weak inhibitors of OCT2 and OAT1, and CPC is a high affinity substrate of OAT1.
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http://dx.doi.org/10.1016/j.ijpharm.2014.02.003DOI Listing
April 2014

Pharmacokinetic study of luteolin, apigenin, chrysoeriol and diosmetin after oral administration of Flos Chrysanthemi extract in rats.

Fitoterapia 2012 Dec 20;83(8):1616-22. Epub 2012 Sep 20.

Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.

Flos Chrysanthemi (the flower of Chrysanthemum morifolium Ramat.) is widely used in China as a food and traditional Chinese medicine for many diseases. Luteolin and apigenin are two main bioactive components in Flos Chrysanthemi, and chrysoeriol and diosmetin are two methylated metabolites of luteolin in vivo by cathechol-O-methyltransferase (COMT). However, there was lack of pharmacokinetic information of chrysoeriol and diosmetin after oral administration of Flos Chrysanthemi extract (FCE). The present study aimed to develop an HPLC-UV method for simultaneous determination of rat plasma concentration of luteolin, apigenin, chrysoeriol and diosmetin and utilize it in pharmacokinetic study of the four compounds after orally giving FCE to rats. The method was successfully validated and applied to the pharmacokinetic study when oral administration of FCE to rats with or without co-giving a COMT inhibitor, entacapone. Chrysoeriol and diosmetin were detected in rat plasma after oral administration of FCE and their concentrations were significantly decreased after co-giving entacapone. Furthermore, AUC of luteolin was significantly increased by entacapone, while that of chrysoeriol was decreased by entacapone, which revealed COMT might play an important role in the disposition of luteolin in rats after dosing of FCE. In conclusion, a sensitive, accurate and reproducible HPLC-UV method for simultaneous determination of luteolin, apigenin, chrysoeriol and diosmetin in rat plasma were developed, pharmacokinetics of chrysoeriol and diosmetin combined with luteolin and apigenin were characterized after oral administration of FCE to rats, which gave us more information on pharmacokinetics and potential pharmacological effects of FCE in vivo.
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http://dx.doi.org/10.1016/j.fitote.2012.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127355PMC
December 2012
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