Publications by authors named "Zhaoyuan Chen"

17 Publications

  • Page 1 of 1

NLK suppresses MAVS-mediated signaling in black carp antiviral innate immunity.

Dev Comp Immunol 2021 Apr 17;122:104105. Epub 2021 Apr 17.

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China. Electronic address:

Mammalian Nemo-like kinase (NLK) plays important roles in multiple biological processes including immune response; however, the roles of teleost NLK remain largely unknown. In the present study, the NLK homolog (bcNLK) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The coding region of bcNLK consists of 1427 nucleotides and encodes 476 amino acid, including two low complexity region (LCR) domains at the N-terminus and a serine/threonine protein kinase catalytic (S-TKc) domain in the middle region. The transcription of bcNLK are promoted after spring viremia of carp virus (SVCV) infection and poly (I:C) stimulation in host cells, but not post LPS treatment. bcNLK exhibits weak impact on the transcription of interferon (IFN) promoter in the reporter assay, however, black carp MAVS (bcMAVS)-mediated IFN promoter transcription is remarkably dampened by bcNLK. The interaction between bcNLK and bcMAVS is detected through the co-immunoprecipitation assay. Accordingly, the plaque assay results show that bcMAVS-mediated antiviral ability is impaired by bcNLK. Moreover, knockdown of bcNLK in host cells leads to the enhanced antiviral ability against SVCV. All these data support the conclusion that black carp NLK associates with MAVS and inhibited MAVS-mediated antiviral signaling.
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http://dx.doi.org/10.1016/j.dci.2021.104105DOI Listing
April 2021

Review: The Emerging Role of Neutrophil Extracellular Traps in Sepsis and Sepsis-Associated Thrombosis.

Front Cell Infect Microbiol 2021 17;11:653228. Epub 2021 Mar 17.

Department of Anesthesiology, Zhongshan Hospital, Fudan University, Shanghai, China.

Patients with sepsis commonly suffer from coagulation dysfunction and lead to the formation of thrombus. During the development of sepsis, neutrophils migrate from the circulating blood to infected tissues and mediate the formation of neutrophil extracellular traps (NETs) that kill pathogens. However, the overactivation of neutrophils can promote the formation of immunothrombosis and even cause disseminated intravascular coagulation (DIC), which damages microcirculation. The outcome of sepsis depends on early recognition and intervention, so clinical evaluation of NETs function may be a valuable biomarker for early diagnosis of sepsis. The interaction of NETs with platelets, complement, and endothelium mediates the formation of immunothrombosis in sepsis. Inhibiting the formation of NETs is also considered to be one of the potential treatments for sepsis. In this review, we will discuss the key role of neutrophils and NETs in sepsis and septic thrombosis, in order to reveal new mechanisms for thrombosis treatment of sepsis.
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http://dx.doi.org/10.3389/fcimb.2021.653228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010653PMC
March 2021

TRIF-mediated antiviral signaling is differentially regulated by TRAF2 and TRAF6 in black carp.

Dev Comp Immunol 2021 Mar 22;121:104073. Epub 2021 Mar 22.

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China. Electronic address:

TRIF is an antiviral adaptor downstream of Toll-like receptors, the roles of teleost TRIF and their regulation remain largely unknown. In this study, a TRIF homologue (bcTRIF) of black carp (Mylopharyngodon piceus) has been cloned, and the transcription of bcTRIF in vivo and ex vivo increased in response to different stimuli. Overexpressed bcTRIF induced the transcription of interferon promoter in the EPC cells and enhanced protection of cells against infection of spring viremia of carp virus (SVCV). The previous study has identified that black carp TRAF2 (bcTRAF2) and TRAF6 (bcTRAF6) functioned positively in RIG-I/MAVS signaling. When co-expressed with bcTRAF2, bcTRIF-induced the transcription of interferon promoter in EPC cells was decreased, and the antiviral activity of bcTRIF was dampened accordingly. On the contrary, co-expressed bcTRAF6 enhanced both bcTRIF-mediated interferon promoter transcription and antiviral activity. The subsequent co-immunoprecipitation identified the interaction between bcTRAF2/6 and bcTRIF. Thus, bcTRIF-mediated antiviral signaling is up-regulated by bcTRAF6 and down-regulated by bcTRAF2.
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http://dx.doi.org/10.1016/j.dci.2021.104073DOI Listing
March 2021

Black carp TRADD suppresses MAVS/IFN signaling during the innate immune activation.

Fish Shellfish Immunol 2021 Apr 26;111:83-93. Epub 2021 Jan 26.

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China. Electronic address:

Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and up-regulates MAVS/IFN signaling pathway in human and mammal. However, the role of TRADD in teleost fish remains obscure. To reveal the function of teleost TRADD in the innate immune response, the TRADD homologue (bcTRADD) of black carp (Mylopharyngodon piceus) has been cloned and the function of bcTRADD is investigated in this study, which shares similar functional domain to its mammalian counterpart. bcTRADD mRNA expression level increased in response to different stimuli, including LPS, poly (I:C) and virus infection in host cells. bcTRADD activated the transcriptional activity of NF-κB promoter in the reporter assay; however, showed hardly any effect on the transcriptional activity of IFN promoter. It was interesting that black carp mitochondria antiviral signaling protein (bcMAVS)-activated IFN promoter transcription were dramatically depressed by bcTRADD and the C-terminal death domain of bcTRADD was indispensable for its regulation of bcMAVS. Accordingly, the plaque assay result showed that EPC cells co-expressing bcMAVS and bcTRADD presented much attenuated antiviral activity than EPC cells expressing bcMAVS alone. Knockdown of bcTRADD slightly promoted the antiviral ability of the host cells against SVCV. The current data support the conclusion that bcTRADD suppresses MAVS-mediated antiviral signaling, which is different to its mammalian counterpart.
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http://dx.doi.org/10.1016/j.fsi.2021.01.006DOI Listing
April 2021

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids.

Bioorg Med Chem 2020 12 11;28(23):115785. Epub 2020 Oct 11.

Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, United States.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.
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http://dx.doi.org/10.1016/j.bmc.2020.115785DOI Listing
December 2020

Black carp RIPK1 negatively regulates MAVS-mediated antiviral signaling during the innate immune activation.

Dev Comp Immunol 2020 08 4;109:103726. Epub 2020 May 4.

State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha, 410081, China. Electronic address:

Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that black carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.
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http://dx.doi.org/10.1016/j.dci.2020.103726DOI Listing
August 2020

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids.

Bioorg Med Chem 2018 05 21;26(9):2271-2279. Epub 2018 Feb 21.

Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591, United States.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.
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http://dx.doi.org/10.1016/j.bmc.2018.02.025DOI Listing
May 2018

Molecular Insights into Hydrogen Peroxide-sensing Mechanism of the Metalloregulator MntR in Controlling Bacterial Resistance to Oxidative Stresses.

J Biol Chem 2017 03 21;292(13):5519-5531. Epub 2017 Feb 21.

From the State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Chaoyang District, Beijing 100101, China,

Manganese contributes to anti-oxidative stress particularly in catalase-devoid bacteria, and DtxR family metalloregulators, through sensing cellular Mn content, regulate its homeostasis. Here, we show that metalloregulator MntR (So-MntR) functions dually as Mn and HO sensors in mediating HO resistance by an oral streptococcus. HO disrupted So-MntR binding to Mn transporter promoter and induced disulfide-linked dimerization of the protein. Mass spectrometry identified Cys-11/Cys-156 and Cys-11/Cys-11 disulfide-linked peptides in HO-treated So-MntR. Site mutagenesis of Cys-11 and Cys-156 and particularly Cys-11 abolished HO-induced disulfide-linked dimers and weakened HO damage on So-MntR binding, indicating that HO inactivates So-MntR via disulfide-linked dimerization. So-MntR C123S mutant was extremely sensitive to HO oxidization in dimerization/oligomerization, probably because the mutagenesis caused a conformational change that facilitates Cys-11/Cys-156 disulfide linkage. Intermolecular Cys-11/Cys-11 disulfide was detected in C123S/C156S double mutant. Redox Western blot detected So-MntR oligomers in air-exposed cells but remarkably decreased upon HO pulsing, suggesting a proteolysis of the disulfide-linked So-MntR oligomers. Remarkably, elevated C11S and C156S but much lower C123S proteins were detected in HO-pulsed cells, confirming Cys-11 and Cys-156 contributed to HO-induced oligomerization and degradation. Accordingly, in the C11S and C156S mutants, expression of and cellular Mn decreased, but HO susceptibility increased. In the C123S mutant, increased expression, cellular Mn content, and manganese-mediated HO survival were determined. Given the wide distribution of Cys-11 in streptococcal DtxR-like metalloregulators, the disclosed redox regulatory function and mechanism of So-MntR can be employed by the DtxR family proteins in bacterial resistance to oxidative stress.
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http://dx.doi.org/10.1074/jbc.M116.764126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392694PMC
March 2017

Alternative formulations of sorafenib for use in children.

Pediatr Blood Cancer 2013 Oct 20;60(10):1642-6. Epub 2013 Jun 20.

Department of Oncology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.

Background: Sorafenib is an oral multikinase inhibitor with antiangiogenic and antitumor activity. In most cases, the commercially available 200 mg tablet is not suitable for administration to children. We studied the chemical and physical stability of extemporaneously prepared formulations and evaluated the pharmacokinetic profile of cut tablets and smaller-dosage capsules of sorafenib in children.

Procedure: Commercially available 200 mg tablets of sorafenib tosylate were used to prepare liquid suspensions of sorafenib in oil and Ora-Plus(®):Ora-Sweet(®) solution, and to prepare 5, 10, 20, 50, and 100 mg capsules. Plasma concentrations of sorafenib were measured in patients receiving capsules and cut tablets, using a validated HPLC-based method with tandem mass spectrometric detection.

Results: At room temperature and under refrigeration, sorafenib concentrations in Ora Plus(®):Ora Sweet(®) were highly variable (means ranging from 75% to 131% of the intended concentration of 50 mg/ml). In oil suspension, sorafenib concentrations were inconsistent during compounding. In contrast, all smaller-dosage capsules, except the 5 mg capsule, were within 91-99% of the intended content and were stable at room temperature for at least 8 months. Sorafenib pharmacokinetic parameters in patients receiving capsules or cut tablets were consistent with those reported previously in adults and children receiving intact tablets.

Conclusions: Sorafenib is not stable in an oral suspension prepared from commercially available tablets, but compounded capsules in smaller-dosage forms that can be sprinkled on food or cut tablets are alternatives for administration to children who need smaller doses based on body surface area or cannot swallow tablets.
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http://dx.doi.org/10.1002/pbc.24619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3800690PMC
October 2013

Sensitive, accurate and simple liquid chromatography-tandem mass spectrometric method for the quantitation of amphotericin B in human or minipig plasma.

J Chromatogr Sci 2012 Aug 4;50(7):636-43. Epub 2012 May 4.

Analytical Pharmacology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.

Amphotericin B (AMB) is still the standard care for systemic fungal infections. This paper describes a sensitive, accurate and simple liquid chromatography-tandem mass spectrometry method to quantify AMB in human or minipig plasma. Samples were prepared through protein precipitation by adding methanol-acetonitrile (1:3, v/v) to either human or minipig plasma. High-performance liquid chromatography separation was conducted on a 10-cm Gemini C18 column with a 7-min gradient of mobile phase comprised of buffer A (0.1% formic acid aqueous solution) and buffer B (methanol-acetonitrile, 2:3, v/v). AMB was detected through multiple reaction monitoring (MRM) with a mass transition of 924.60 → 743.30 and the internal standard paclitaxel was detected through MRM with a mass transition of 854.30 → 286.10. The method had a linear range between 5 and 2500 ng/mL with lower limit of quantitation of 3 ng/mL. The overall recovery was 113 ± 4.06% in human plasma and 94.8 ± 7.38% in minipig plasma. The method has been validated and applied for AMB pharmacokinetic study in both human and minipig plasma.
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http://dx.doi.org/10.1093/chromsci/bms049DOI Listing
August 2012

Inhibition of OCTN2-mediated transport of carnitine by etoposide.

Mol Cancer Ther 2012 Apr 2;11(4):921-9. Epub 2012 Mar 2.

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

OCTN2 is a bifunctional transporter that reabsorbs filtered carnitine in a sodium-dependent manner and secretes organic cations into urine as a proton antiport mechanism. We hypothesized that inhibition of OCTN2 by anticancer drugs can influence carnitine resorption. OCTN2-mediated transport inhibition by anticancer drugs was assessed using cells transfected with human OCTN2 (hOCTN2) or mouse Octn2 (mOctn2). Excretion of carnitine and acetylcarnitine was measured in urine collected from mice and pediatric patients with cancer before and after administration of etoposide. Five of 27 tested drugs (50-100 μmol/L) inhibited hOCTN2-mediated carnitine uptake by 42% to 85% (P < 0.001). Of these inhibitors, etoposide was itself a transported substrate of hOCTN2 and mOctn2. Etoposide uptake by hOCTN2 was reversed in the presence of excess carnitine. This competitive inhibitory mechanism was confirmed in an in silico molecular docking analysis. In addition, etoposide inhibited the transcellular apical-to-basolateral flux of carnitine in kidney cells. Etoposide was also associated with a significant urinary loss of carnitine in mice (~1.5-fold) and in patients with cancer (~2.4-fold). Collectively, these findings indicate that etoposide can inhibit hOCTN2 function, potentially disturb carnitine homeostasis, and that this phenomenon can contribute to treatment-related toxicities.
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http://dx.doi.org/10.1158/1535-7163.MCT-11-0980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3466062PMC
April 2012

Cisplatin-induced downregulation of OCTN2 affects carnitine wasting.

Clin Cancer Res 2010 Oct 21;16(19):4789-99. Epub 2010 Sep 21.

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Purpose: Carnitine is an essential cofactor for mitochondrial fatty acid oxidation that is actively reabsorbed by the luminal transporter Octn2 (Slc22a5). Because the nephrotoxic agent cisplatin causes urinary loss of carnitine in humans, we hypothesized that cisplatin may affect Octn2 function.

Experimental Design: Excretion of carnitine and acetylcarnitine was measured in urine collected from mice with or without cisplatin administration. The transport of carnitine was assessed in cells that were transfected with OCT1 or OCT2. The effect of cisplatin treatment on gene expression was analyzed using a mouse GeneChip array and validated using quantitative reverse transcriptase-PCR.

Results: In wild-type mice, urinary carnitine excretion at baseline was ∼3-fold higher than in mice lacking the basolateral cisplatin transporters Oct1 and Oct2 [Oct1/2(-/-) mice], indicating that carnitine itself undergoes basolateral uptake into the kidney. Transport of carnitine by OCT2, but not OCT1, was confirmed in transfected cells. We also found that cisplatin caused an increase in the urinary excretion of carnitine and acetylcarnitine in wild-type mice but not in Oct1/2(-/-) mice, suggesting that tubular transport of cisplatin is a prerequisite for this phenomenon. Cisplatin did not directly inhibit the transport of carnitine by Octn2 but downregulated multiple target genes of the transcription factor peroxisome proliferator activated receptor α, including Slc22a5, in the kidney of wild-type mice that were absent in Oct1/2(-/-) mice.

Conclusion: Our study shows a pivotal role of Oct1 and Oct2 in cisplatin-related disturbances in carnitine homeostasis. We postulate that this phenomenon is triggered by deactivation of peroxisome proliferator activated receptor α and leads to deregulation of carnitine-shuttle genes.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-1239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531239PMC
October 2010

Marginal increase of sunitinib exposure by grapefruit juice.

Cancer Chemother Pharmacol 2011 Mar 29;67(3):695-703. Epub 2010 May 29.

Department of Clinical Pharmacy and Toxicology, Leiden University Medical Center, Leiden, The Netherlands.

Purpose: The drug label of sunitinib includes a warning for concomitant use of grapefruit juice (GJ) but clinical evidence for this drug interaction is lacking. The aim of this study is to determine the effect of GJ, a potent intestinal cytochrome P450 (CYP) 3A4 inhibitor, on steady-state sunitinib pharmacokinetics (PK).

Methods: Sunitinib PK was evaluated in eight cancer patients receiving sunitinib monotherapy in a "4 weeks on-2 weeks off" dose regimen. Serial blood samples for PK analysis of sunitinib were collected on two separate days. On both PK days, patients received a single oral dose of 7.5-mg midazolam as a phenotypic probe for assessment of intestinal CYP3A4 activity. The first PK day was at steady-state sunitinib PK (between days 14-20), the second PK day was on day 28. On days 25, 26 and 27, 200-mL GJ was consumed 3 times a day. The effect of GJ on sunitinib exposure was assessed by comparing sunitinib PK with and without GJ.

Results: Concomitant use of GJ and sunitinib resulted in an 11% increase of the relative bioavailability of sunitinib (P < 0.05). The effect of GJ on CYP3A4 activity was confirmed by an increase of ~50% of mean midazolam exposure (AUC(0-24 h)) from 122.1 to 182.0 ng h/mL (P = 0.034).

Conclusion: GJ consumption results in a marginal increase in sunitinib exposure which is not considered clinically relevant. There is no clinical evidence underscoring the warning in the sunitinib drug label regarding concomitant use of GJ.
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http://dx.doi.org/10.1007/s00280-010-1367-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3043256PMC
March 2011

Interaction of the multikinase inhibitors sorafenib and sunitinib with solute carriers and ATP-binding cassette transporters.

Clin Cancer Res 2009 Oct 22;15(19):6062-9. Epub 2009 Sep 22.

Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

Purpose: To compare side-by-side the uptake of sorafenib and sunitinib in vitro by human uptake solute carriers of the SLC22A and SLCO families, the transport by and inhibition of efflux ATP-binding cassette (ABC) transporters, and the role of ABCB1 in the plasma pharmacokinetics and brain penetration of these agents.

Experimental Design: Uptake of [(3)H]sorafenib or [(3)H]sunitinib was assessed in Xenopus laevis oocytes or mammalian cells transfected with cDNAs coding for human OATP1A2, OATP1B1, OATP1B3, OCT1, OAT2, OAT3, OCTN1, or OCTN2. Efflux and inhibition experiments were conducted in cells transfected with human ABCB1, ABCG2, ABCC2, or ABCC4. In vivo pharmacokinetic studies were done in knockout mice lacking Abcb1-type transporters.

Results: Intracellular uptake was not appreciably affected by any of the studied solute carriers and was minute relative to the respective prototypical substrates. Sorafenib and sunitinib showed concentration-dependent (1 and 10 micromol/L), low to moderate affinity for ABCB1 but were not affected by the other ABC transporters. Both agents inhibited all tested ABC transporters. The absence of Abcb1 had no affect on plasma pharmacokinetics, but brain penetration was moderately increased by 1.9- and 2.9-fold for sorafenib and sunitinib, respectively, in knockout animals versus controls.

Conclusions: Unlike other tyrosine kinase inhibitors, sorafenib and sunitinib do not appear to rely on active transport to enter the cell nor are they high-affinity substrates for ABC efflux transporters. Based on these characteristics, these two drugs may be less susceptible to transporter-mediated alterations in systemic exposure and transporter-related resistance mechanisms.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-0048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774722PMC
October 2009

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2008 Oct 12;874(1-2):84-8. Epub 2008 Sep 12.

Pharmaceutical Sciences Department, St. Jude Children's Research Hospital, Memphis, TN, USA.

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.
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http://dx.doi.org/10.1016/j.jchromb.2008.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612784PMC
October 2008

Efficient polymer monolith for strong cation-exchange capillary liquid chromatography of peptides.

Anal Chem 2006 Jun;78(11):3509-18

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602-5700, USA.

A stable poly[2-acrylamido-2-methyl-1-propanesulfonic acid-co-poly(ethylene glycol) diacrylate] monolith was synthesized inside a 75-microm-i.d. capillary by photoinitiated copolymerization with water, methanol, and ethyl ether as porogens. The resulting monolith was evaluated for strong cation-exchange capillary liquid chromatography of both synthetic and natural peptides. Although the monolith possessed relatively strong hydrophobicity due to the use of 2-acrylamido-2-methyl-1-propanesulfonic acid as one monomer, the monolith had a high dynamic binding capacity of 157 microequiv of peptide/mL, or 332 mg of cytochrome c/mL. Exceptionally high resolution resulting from extremely narrow peaks was obtained, resulting in a peak capacity of 179 when using a shallow salt elution gradient. Although a second, naturally formed gradient might contribute to the sharp peaks obtained, high efficiency was mainly due to the use of poly(ethylene glycol) diacrylate as a biocompatible cross-linker.
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http://dx.doi.org/10.1021/ac060284rDOI Listing
June 2006

Initial analysis of the phosphoproteome of Chinese hamster ovary cells using electrophoresis.

J Biomol Tech 2004 Dec;15(4):249-56

Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.

Protein phosphorylation is a common post-translational modification of enormous biological importance. Analysis of phosphorylation at the global level should shed light on the use of this modification to regulate metabolism, signal transduction, and other processes. We have begun a proteomic analysis of phosphorylation using two-dimensional gel electrophoresis. Chinese hamster ovary (CHO) cells were metabolically labeled using 32P-orthophosphate. The proteins were extracted and run on two-dimensional electrophoresis. Gels were stained using colloidal Coomassie stain, dried, and phosphorimaged. The Coomassie stain allowed the observation of 468 individual protein spots. The phosphorimage showed 181 spots. The phosphoproteome of CHO cells therefore comprises around one third as many proteins as the CHO cell abundance proteome. However, the most intense spots in the phosphoproteome usually do not correlate with intense spots in the abundance proteome. We investigated the effects of labeling time, finding that the number of observable spots increases but the relative intensities also change. We also investigated the effects of adding a phosphatase inhibitor during labeling. Finally, we evaluated a phosphoprotein-specific stain (Pro-Q Diamond) in comparison with radiolabeling methods. There is not perfect correlation between radiolabeled phosphoproteins and Pro-Q Diamond-stained phosphoproteins.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291695PMC
December 2004