Interspecies variability in expression of hepatobiliary

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Running Title Interspecies variability in hepatobiliary transporters expression. Corresponding author Jashvant D Unadkat Department of Pharmaceutics University of. Washington Seattle P O Box 357610 WA 98195 USA Phone 1 206 685 2869 Fax 1. 206 543 3204,Email jash u washington edu, Number of text pages 29 including tables figure legends and references. Number of tables 2,Number of figures 3,Number of supplement tables figures 4. Number of references 31,Number of words in the Abstract 240. Number of words in the Introduction 707, ABBREVIATIONS DNP SG 2 4 dinitrophenyl S glutathione BCRP breast cancer. resistance protein BSEP bile salt export pump CE collision energy CMVs canalicular. membrane vesicles D beagle dog FDA the Food and Drug Administration LC MS MS liquid. chromatography tandem mass spectrometry LLOQ lower limit of quantification kcat turnover. number Km Michaelis constant mRNA messenger RNA M cynomolgus monkey MDR1. multidrug resistance MRP multidrug resistance associated protein NTCP sodium taurocholate. cotransporting polypeptide OATP organic anion transporting polypeptide OCT1 organic. cation transporter 1 PCR polymerase chain reaction PK pharmacokinetic RT retention time. QC quality control SD Sprague Dawley rat SIL stable isotope labeled W Wistar rat SNP. single nucleotide polymorphism Vmax maximal velocity. We quantified by LC MS MS transporter protein expression of BSEP MATE1 MRP3 MRP4. NTCP and OCT1 in our human liver bank n 55 and determined the relationship between. protein expression and sex age and genotype These data complement our previous work in the. same liver bank where we quantified the protein expression of OATPs BCRP MDR1 and. MRP2 In addition we quantified and compared the interspecies differences in expression of the. hepatobiliary transporters corresponding to the above human transporters in liver tissue and. hepatocytes of male beagle dogs cynomolgus monkeys Sprague Dawley and Wistar rats In all. the species the sinusoidal OATPs Oatps were the most abundant hepatic transporters. However there were notable interspecies differences in the relative abundance of the remaining. transporters For example the next most abundant transporter in humans and monkeys was. OCT1 Oct1 while it was Mrp2 and Ntcp in dogs Wistar rats and Sprague Dawley rats. respectively In contrast the protein expression of the efflux transporters BCRP Bcrp. MDR1 Mdr1 MRP3 Mrp3 MRP4 Mrp4 MATE1 Mate1 was much lower across all the species. For most transporters the expression in the liver tissues was comparable to that in the unplated. cryopreserved hepatocytes These data on human liver transporter protein expression complete. the picture of the expression of major human hepatobiliary transporters important in drug. disposition and toxicity In addition the data on expression of the corresponding hepatobiliary. transporters in preclinical species will be helpful in interpreting and extrapolating. pharmacokinetic pharmacological and toxicological results from pre clinical studies to humans. Introduction, Hepatotoxicity is a major cause of failure in drug development Kaplowitz 2001 During drug.
development the FDA requires in vivo repeated dose toxicity studies of a drug candidate to be. conducted in at least two animal species one of which must be a non rodent before initiating. human studies, http www fda gov downloads drugs guidancecomplianceregulatoryinformation guidances ucm. 292340 pdf Typically these species are mouse or rat and dog or monkey In order to interpret. hepatotoxicity data generated in animals and predict their clinical relevance it is important to. understand the potential interspecies differences in mechanisms of toxicity of drugs One such. mechanism is transport of drugs into and out of the liver which can result in interspecies. differences in hepatic concentration of the drug Lai 2009 For example a drug may be rapidly. cleared from the liver in one animal species because of high expression of a hepatobiliary. transporter As a result the hepatic concentrations of the drug will be low In contrast another. species which has much lower expression of the same transporter may demonstrate elevated. hepatic concentration of the drug potentially causing hepatotoxicity Therefore it is important. to compare the expression of transporters in liver tissue of humans and animals routinely used in. toxicological studies In addition allometric scaling has been successfully used to predict. human pharmacokinetics PK based on studies conducted in preclinical species Huang and. Riviere 2014 However these methods generally perform poorly for transporter substrates due. to species differences in the substrate specificity tissue distribution and relative abundance of. transporters Chu et al 2013 Quantitative knowledge of species differences in transporters. especially at the protein and functional level will be useful to fill this knowledge gap and help. improve allometric scaling of PK and drug drug interactions from preclinical species to humans. Thus far interspecies differences in expression of transporters have mostly been compared by. quantifying the expression of the mRNA of the transporter by quantitative PCR or qPCR or. protein by Western blotting in the tissues of species of interest However both methods have. shortcomings Often for transporters the mRNA expression does not correlate well with protein. expression For example we and others have shown that mRNA expression of transporters is. not always correlated with protein amounts in human livers Ohtsuki et al 2012 Prasad et al. 2013 Western blotting has the disadvantage that it is semi quantitative and in the absence of. pure protein standards does not allow comparison of expression across proteins or species due to. differences in affinity of the antibodies used Transporter protein quantification using surrogate. signature peptides and liquid chromatography mass spectrometry LC MS MS overcomes these. limitations Li et al 2009a Li et al 2009b Ito et al 2011 Ohtsuki et al 2012 Groer et al. 2013 Prasad et al 2013 Prasad et al 2014 Briefly enzymatic digestion commonly by. trypsin is performed on target transporter protein and one or more of the liberated unique. signature peptides is quantified by LC MS MS The corresponding synthetic peptide as well as. heavy labeled peptide is used as the calibrator and internal standard respectively This method. allows simultaneous measurement of multiple transporters and if there is sequence identity the. same signature peptide s can be used to quantify the same transporter protein in other species. Although the expression of several transporters MRP2 Mrp2 BCRP Bcrp NTCP Ntcp. MDR1 Mdr1 BSEP Bsep in human and animal liver tissues and hepatocytes have been. compared using this approach this comparison did not include all relevant hepatic drug. transporters or animal species studied here Li et al 2009b Li et al 2009c Qiu et al 2013. In the present study we have quantified the protein concentration of BSEP MATE1 MRP3. MRP4 NTCP and OCT1 in human livers n 55 This extends our previous work where we. quantified several important human hepatic drug transporters namely BCRP MDR1 MRP2. and OATPs in the same human liver bank Deo et al 2012 Prasad et al 2013 Prasad et al. 2014 The clinical relevance of these transporters has been summarized in recent reviews by the. International Transporter Consortium Giacomini et al 2010 Hillgren et al 2013 In addition. we have compared the expression of the above human hepatic transporters with those in animals. namely Bcrp Bsep Mdr1 Mrp2 Mrp3 Mrp4 Ntcp Oatps and Oct1 in liver tissues and. unplated cryopreserved hepatocytes from male beagle dogs cynomolgus monkeys and Sprague. Dawley Wistar rats,Materials and Methods, Chemicals and Reagents Synthetic signature peptides for quantified transporters Table 1. were obtained from New England Peptides Boston MA The corresponding stable isotope. labeled SIL internal standards were purchased from Thermo Fisher Scientific Rockford IL. The ProteoExtract native membrane protein extraction kit was purchased from Calbiochem. Temecula CA Ammonium bicarbonate 98 purity and sodium deoxycholate 98 purity. were obtained from Thermo Fisher Scientific Rockford IL and MP Biomedicals Santa Ana. CA respectively BCA protein assay and the in solution trypsin digestion kit were obtained. from Pierce Biotechnology Rockford IL HPLC grade acetonitrile 99 9 purity methanol. 99 9 purity and formic acid 99 5 purity were purchased from Fischer Scientific Fair. Lawn NJ Deionized water was generated from a Q Gard 2 Purification Pack water purifying. system Millipore Bedford MA Iodoacetamide and dithiothreitol were obtained from Pierce. Biotechnology Rockford IL, Liver Samples and Hepatocytes Fifty five liver tissue samples from the human liver bank of. the School of Pharmacy University of Washington were used Procurement approved by the. University of Washington Human Subjects Division characteristics and storage of these. samples have been previously described Paine et al 1997 with additional details reported by. Prasad et al Prasad et al 2014 The subjects were Caucasian n 51 one Asian male and. three black males not of hispanic origin Subject age range was 9 to 70 years and comprised of. 29 females and 26 males The majority of the livers were obtained from organ donors who met. with accidental death e g trauma from vehicular accidents subarachnoid hemorrhage. cerebrovascular accident and were harvested from breathing donors who were perfused with. University of Wisconsin solution for organ transplant purposes Six male beagle dog and six. male Sprague Dawley rat liver tissue samples were obtained from Bioreclamation IVT. Hicksville NY Ten male cynomolgus monkey liver tissue samples from Indonesian origin. were provided by Merck White House Station NJ Cryopreserved hepatocytes from three male. beagle dogs and five male cynomolgus monkeys were purchased from Life Technologies. Carlsbad CA Cryopreserved hepatocytes from three Sprague Dawley rats were obtained from. Triangle Research Labs Charlottesville VA Ten male liver tissue samples and one batch of. cryopreserved hepatocytes from Wistar rats were provided by AstraZeneca Boston MA. Peptide Selection Peptide sequences of the transporter proteins were obtained from two. databases Uniprot and Pubmed Transmembrane helices in transporter proteins were predicted. using two different methods TMHMM Krogh et al 2001 and Phobius Kall et al 2007. Whenever possible two unique signature peptides not present in any other known proteins were. selected for quantification of each transporter Table 1 based on previously reported criteria. Kamiie et al 2008 Prasad and Unadkat 2014 Briefly the selected peptides were between 9. and 16 amino acid residues not embedded in the membrane or containing any known. polymorphic variations or posttranslational modifications In addition peptides susceptible to. degradation e g containing methionine or cysteine residues were not selected Continuous R. and K sequences RR RK KR and KK were excluded to avoid miscleavages by trypsin. Whenever possible we used the same signature peptides to quantify transporter protein. expression in multiple species see Table 1, Sample Preparation Total membrane protein in triplicate was isolated from liver tissue or. unplated cryopreserved hepatocytes as described previously Prasad et al 2014 Then one. hundred L of 2 0 mg mL or lower concentration of liver tissue or hepatocytes membrane. protein were incubated with 20 L of dithiothreitol 100 mM and 50 L of ammonium. bicarbonate buffer 50 mM pH 7 8 After incubation at 95 C for 5 min 20 L of. iodoacetamide 200 mM an alkylating agent were added to the mixture followed by incubation. at room temperature for 20 min in the dark To concentrate the sample ice cold methanol 0 5. mL chloroform 0 2 mL and water 0 2 mL were added to each sample After centrifugation. at 4 C for 5 min at 16 000 g the pellet was washed once with ice cold methanol 0 5 mL and. was resuspended with 40 L of reconstitution solution mixture of equal volume of 3 sodium. deoxycholate w v and 50 mM ammonium bicarbonate buffer Finally the protein sample was. digested with 20 L of trypsin Protein to trypsin ratio was 25 1 w w After 24 h incubation at. 37 C the digestion reaction was quenched by 30 L of labeled peptide internal standard SIL. cocktail prepared in 50 acetonitrile in water containing 0 1 formic acid final concentration. of SIL 0 1 0 25 nM The samples were centrifuged at 5000g for 5 min at 4 C and 5 L of the. supernatant was introduced into the LC MS MS system. The calibrators in duplicate were prepared by spiking peptide standards into the extraction. buffer II of the membrane protein extraction kit The quality control QC samples were. prepared in triplicate by spiking peptides into extraction buffer II three concentrations at low. medium and high level of the calibration curve or pooled liver membrane two concentrations. at medium and high level of the calibration curve, Liquid Chromatography tandem Mass Spectroscopy Analyses Waters Xevo TQS tandem.
mass spectrometer coupled to Waters Acquity UPLC system Waters Hertfordshire UK. operated in electrospray positive ionization mode was used for liquid chromatography tandem. mass spectroscopy LC MS MS analyses of the signature peptides The mass spectrometry. conditions were as follows capillary 3 5 kV source offset 30 V source temperature 500 C see. Interspecies variability in expression of hepatobiliary transporters across human dog monkey and rat as determined by quantitative proteomics Li Wang Bhagwat Prasad Laurent Salphati Xiaoyan Chu Anshul Gupta Cornelis E C A Hop Raymond Evers and Jashvant D Unadkat

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