Integrative molecular characterisation of gallbladder cancer reveals micro-environment-associated subtypes.

BACKGROUND & AIMS: Gallbladder cancer (GBC) is the most common type of biliary tract cancer, but the molecular mechanisms involved in gallbladder carcinogenesis remain poorly understood. In this study, we applied integrative genomics approaches to characterise GBC and explore molecular subtypes associated with patient survival. METHODS: We profiled the mutational landscape of GBC tumours (whole-exome sequencing on 92, targeted sequencing on 98, in total 190 patients). In a subset (n = 45), we interrogated the matched transcriptomes, DNA methylomes, and somatic copy number alterations. We explored molecular subtypes identified through clustering tumours by genes whose expression was associated with survival in 47 tumours and validated subtypes on 34 publicly available GBC cases. RESULTS: Exome analysis revealed TP53 was the most mutated gene. The overall mutation rate was low (median 0.82 Mut/Mb). APOBEC-mediated mutational signatures were more common in tumours with higher mutational burden. Aflatoxin-related signatures tended to be highly clonal (present in ≥50% of cancer cells). Transcriptome-wide survival association analysis revealed a 95-gene signature that stratified all GBC patients into 3 subtypes that suggested an association with overall survival post-resection. The 2 poor-survival subtypes were associated with adverse clinicopathologic features (advanced stage, pN1, pM1), immunosuppressive micro-environments (myeloid-derived suppressor cell accumulation, extensive desmoplasia, hypoxia) and T cell dysfunction, whereas the good-survival subtype showed the opposite features. CONCLUSION: These data suggest that the tumour micro-environment and immune profiles could play an important role in gallbladder carcinogenesis and should be evaluated in future clinical studies, along with mutational profiles. LAY SUMMARY: Gallbladder cancer is highly fatal, and its causes are poorly understood. We evaluated gallbladder tumours to see if there were differences between tumours in genetic information such as DNA and RNA. We found evidence of aflatoxin exposure in these tumours, and immune cells surrounding the tumours were associated with survival.

In Vivo-to-In Vitro Extrapolation of Transporter-Mediated Renal Clearance: Relative Expression Factor Versus Relative Activity Factor Approach.

About 30% of approved drugs are cleared predominantly by renal clearance (CLr). Of these, many are secreted by transporters. For these drugs, in vitro-to-in vivo extrapolation of transporter-mediated renal secretory clearance (CLsec,plasma) is important to prospectively predict their renal clearance and to assess the impact of drug-drug interactions and pharmacogenetics on their pharmacokinetics. Here we compared the ability of the relative expression factor (REF) and the relative activity factor (RAF) approaches to quantitatively predict the in vivo CLsec,plasma of 26 organic anion transporter (OAT) substrates assuming that OAT-mediated uptake is the rate-determining step in the CLsec,plasma of the drugs. The REF approach requires protein quantification of each transporter in the tissue (e.g., kidney) and transporter-expressing cells, whereas the RAF approach requires the use of a transporter-selective probe substrate (both in vitro and in vivo) for each transporter of interest. For the REF approach, 50% and 69% of the CLsec,plasma predictions were within 2- and 3-fold of the observed values, respectively; the corresponding values for the RAF approach were 65% and 81%. We found no significant difference between the two approaches in their predictive capability (as measured by accuracy and bias) of the CLsec,plasma or CLr of OAT drugs. We recommend that the REF and RAF approaches can be used interchangeably to predict OAT-mediated CLsec,plasma Further research is warranted to evaluate the ability of the REF or RAF approach to predict CLsec,plasma of drugs when uptake is not the rate-determining step. SIGNIFICANCE STATEMENT: This is the first direct comparison of the relative expression factor (REF) and relative activity factor (RAF) approaches to predict transporter-mediated renal clearance (CLr). The RAF, but not REF, approach requires transporter-selective probes and that the basolateral uptake is the rate-determining step in the CLr of drugs. Given that there is no difference in predictive capability of the REF and RAF approach for organic anion transporter-mediated CLr, the REF approach should be explored further to assess its ability to predict CLr when basolateral uptake is not the sole rate-determining step.

Effect of TRPA1 activator allyl isothiocyanate (AITC) on rat dural and pial arteries.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channels may have a role in migraine as some substances known to cause headache activate the channel. In the craniovascular system such activation causes a calcitonin gene-related peptide (CGRP)-dependent increase in meningeal blood flow. TRPA1 channels in the endothelium of cerebral arteries cause vasodilation when activated. The headache preventive substance feverfew inhibits activation of TRPA1 channels. In this study we aim to compare and characterize the effect of the TRPA1 agonist allyl isothiocyanate (AITC) on the diameter of rat dural and pial arteries in vivo. METHODS: The genuine closed-cranial window technique in rats was used to examine changes in dural and pial artery diameter and mean arterial blood pressure (MABP) after intracarotid infusion of AITC. Blockade experiments were performed by intravenous infusion of olcegepant, HC-030031, sumatriptan or capsazepine immediately after infusion of AITC, in four different groups of rats. RESULTS: AITC caused a significant dilation of dural arteries, which was inhibited by HC-030031, olcegepant and sumatriptan, but not by capsazepine. In pial arteries AITC caused a significant dilation, which was not inhibited by any of the pre-treatments, suggesting a poor penetration of the blood-brain barrier or autoregulation due to dimethyl sulfoxide (DMSO) mediated decrease in MABP during HC-030031 infusion. AITC did not cause a significant change in MABP. CONCLUSION: AITC causes dilation of dural arteries via a mechanism dependent on CGRP and TRPA1 that is sensitive to sumatriptan. AITC causes a small but significant dilation of pial arteries.

Ontogeny of Hepatic Sulfotransferases and Prediction of Age-Dependent Fractional Contribution of Sulfation in Acetaminophen Metabolism.

Cytosolic sulfotransferases (SULTs), including SULT1A, SULT1B, SULT1E, and SULT2A isoforms, play noteworthy roles in xenobiotic and endobiotic metabolism. We quantified the protein abundances of SULT1A1, SULT1A3, SULT1B1, and SULT2A1 in human liver cytosol samples (n = 194) by liquid chromatography-tandem mass spectrometry proteomics. The data were analyzed for their associations by age, sex, genotype, and ethnicity of the donors. SULT1A1, SULT1B1, and SULT2A1 showed significant age-dependent protein abundance, whereas SULT1A3 was invariable across 0-70 years. The respective mean abundances of SULT1A1, SULT1B1, and SULT2A1 in neonatal samples was 24%, 19%, and 38% of the adult levels. Interestingly, unlike UDP-glucuronosyltransferases and cytochrome P450 enzymes, SULT1A1 and SULT2A1 showed the highest abundance during early childhood (1 to <6 years), which gradually decreased by approx. 40% in adolescents and adults. SULT1A3 and SULT1B1 abundances were significantly lower in African Americans compared with Caucasians. Multiple linear regression analysis further confirmed the association of SULT abundances by age, ethnicity, and genotype. To demonstrate clinical application of the characteristic SULT ontogeny profiles, we developed and validated a proteomics-informed physiologically based pharmacokinetic model of acetaminophen. The latter confirmed the higher fractional contribution of sulfation over glucuronidation in the metabolism of acetaminophen in children. The study thus highlights that the ontogeny-based age-dependent fractional contribution (fm) of individual drug-metabolizing enzymes has better potential in prediction of drug-drug interactions and the effect of genetic polymorphisms in the pediatric population.

Age- and Genotype-Dependent Variability in the Protein Abundance and Activity of Six Major Uridine Diphosphate-Glucuronosyltransferases in Human Liver.

The ontogeny of hepatic uridine diphosphate-glucuronosyltransferases (UGTs) was investigated by determining their protein abundance in human liver microsomes isolated from 136 pediatric (0-18 years) and 35 adult (age >18 years) donors using liquid chromatography / tandem mass spectrometry (LC-MS/MS) proteomics. Microsomal protein abundances of UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15 increased by ∼8, 55, 35, 33, 8, and 3-fold from neonates to adults, respectively. The estimated age at which 50% of the adult protein abundance is observed for these UGT isoforms was between 2.6-10.3 years. Measured in vitro activity was generally consistent with the protein data. UGT1A1 protein abundance was associated with multiple single nucleotide polymorphisms exhibiting noticeable ontogeny-genotype interplay. UGT2B15 rs1902023 (*2) was associated with decreased protein activity without any change in protein abundance. Taken together, these data are invaluable to facilitate the prediction of drug disposition in children using physiologically based pharmacokinetic modeling as demonstrated here for zidovudine and morphine.

Quantitative characterization of UDP-glucuronosyltransferase 2B17 in human liver and intestine and its role in testosterone first-pass metabolism.

Protein abundance and activity of UGT2B17, a highly variable drug- and androgen-metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC-MS/MS methods. UGT2B17 protein abundance showed >160-fold variation (mean ±â€¯SD, 1.7 ±â€¯2.7 pmol/mg microsomal protein) in adult human liver microsomes (n = 26) and significant correlation (r2 = 0.77, p < 0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (n = 6) showed on average significantly higher protein abundance (7.4 ±â€¯6.6 pmol/mg microsomal protein, p = 0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (n = 5) and enterocytes (n = 16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r2 = 0.69, p = 0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically-based pharmacokinetic (PBPK) models for predicting highly-variable first-pass metabolism of testosterone and other UGT2B17 substrates.

Quantitative Proteomics Reveals Changes in Transporter Protein Abundance in Liver, Kidney and Brain of Mice by Pregnancy.

BACKGROUND: Few studies have systematically investigated pregnancy-induced changes in protein abundance of drug transporters in organs important for drug/xenobiotic disposition. OBJECTIVE: The goal of this study was to compare protein abundance of important drug/xenobiotic transporters including Abcb1a, Abcg2, Abcc2, and Slco1b2 in the liver, kidney and brain of pregnant mice on gestation day 15 to that of non-pregnant mice. METHODS: The mass spectrometry-based proteomics was used to quantify changes in protein abundance of transporters in tissues from pregnant and non-pregnant mice. RESULTS: The protein levels of hepatic Abcc2, Abcc3, and Slco1a4 per µg of total membrane proteins were significantly decreased by pregnancy by 24%, 72%, and 70%, respectively. The protein levels of Abcg2, Abcc2, and Slco2b1 per µg of total membrane proteins in the kidney were significantly decreased by pregnancy by 43%, 50%, and 46%, respectively. After scaling to the whole liver with consideration of increase in liver weight in pregnant mice, the protein abundance of Abcb1a, Abcg2, Abcc2, Abcb11, Abcc4, Slco1a1, and Slco1b2 in the liver was ~50-100% higher in pregnant mice, while those of Abcc3 and Slco1a4 were ~40% lower. After scaling to the whole kidney, none of the transporters examined were significantly changed by pregnancy. Only Abcg2 and Abcb1a were quantifiable in the brain and their abundance in the brain was not influenced by pregnancy. CONCLUSION: Protein abundance of drug transporters can be significantly changed particularly in the liver by pregnancy. These results will be helpful to understand pregnancy-induced changes in drug/xenobiotic disposition in the mouse model.

PBDEs Altered Gut Microbiome and Bile Acid Homeostasis in Male C57BL/6 Mice.

Polybrominated diphenyl ethers (PBDEs) are persistent environmental contaminants with well characterized toxicities in host organs. Gut microbiome is increasingly recognized as an important regulator of xenobiotic biotransformation; however, little is known about its interactions with PBDEs. Primary bile acids (BAs) are metabolized by the gut microbiome into more lipophilic secondary BAs that may be absorbed and interact with certain host receptors. The goal of this study was to test our hypothesis that PBDEs cause dysbiosis and aberrant regulation of BA homeostasis. Nine-week-old male C57BL/6 conventional (CV) and germ-free (GF) mice were orally gavaged with corn oil (10 mg/kg), BDE-47 (100 µmol/kg), or BDE-99 (100 µmol/kg) once daily for 4 days (n = 3-5/group). Gut microbiome was characterized using 16S rRNA sequencing of the large intestinal content in CV mice. Both BDE-47 and BDE-99 profoundly decreased the alpha diversity of gut microbiome and differentially regulated 45 bacterial species. Both PBDE congeners increased Akkermansia muciniphila and Erysipelotrichaceae Allobaculum spp., which have been reported to have anti-inflammatory and antiobesity functions. Targeted metabolomics of 56 BAs was conducted in serum, liver, and small and large intestinal content of CV and GF mice. BDE-99 increased many unconjugated BAs in multiple biocompartments in a gut microbiota-dependent manner. This correlated with an increase in microbial 7α-dehydroxylation enzymes for secondary BA synthesis and increased expression of host intestinal transporters for BA absorption. Targeted proteomics showed that PBDEs downregulated host BA-synthesizing enzymes and transporters in livers of CV but not GF mice. In conclusion, there is a novel interaction between PBDEs and the endogenous BA-signaling through modification of the "gut-liver axis".

Abundance of Phase 1 and 2 Drug-Metabolizing Enzymes in Alcoholic and Hepatitis C Cirrhotic Livers: A Quantitative Targeted Proteomics Study.

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic (n = 27) or hepatitis C (HCV, n = 30) cirrhotic versus noncirrhotic (control) livers (n = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.

Hepatic Abundance and Activity of Androgen- and Drug-Metabolizing Enzyme UGT2B17 Are Associated with Genotype, Age, and Sex.

The major objective of this study was to investigate the association of genetic and nongenetic factors with variability in protein abundance and in vitro activity of the androgen-metabolizing enzyme UGT2B17 in human liver microsomes (n = 455). UGT2B17 abundance was quantified by liquid chromatography-tandem mass spectrometry proteomics, and enzyme activity was determined by using testosterone and dihydrotestosterone as in vitro probe substrates. Genotyping or gene resequencing and mRNA expression were also evaluated. Multivariate analysis was used to test the association of UGT2B17 copy number variation, single nucleotide polymorphisms (SNPs), age, and sex with its mRNA expression, abundance, and activity. UGT2B17 gene copy number and SNPs (rs7436962, rs9996186, rs28374627, and rs4860305) were associated with gene expression, protein levels, and androgen glucuronidation rates in a gene dose-dependent manner. UGT2B17 protein (mean ± S.D. picomoles per milligram of microsomal protein) is sparsely expressed in children younger than 9 years (0.12 ± 0.24 years) but profoundly increases from age 9 years to adults (∼10-fold) with ∼2.6-fold greater abundance in males than in females (1.2 vs. 0.47). Association of androgen glucuronidation with UGT2B15 abundance was observed only in the low UGT2B17 expressers. These data can be used to predict variability in the metabolism of UGT2B17 substrates. Drug companies should include UGT2B17 in early phenotyping assays during drug discovery to avoid late clinical failures.

Critical Issues and Optimized Practices in Quantification of Protein Abundance Level to Determine Interindividual Variability in DMET Proteins by LC-MS/MS Proteomics.

Protein quantification data on drug metabolizing enzymes and transporters (collectively referred as DMET proteins) in human tissues are useful in predicting interindividual variability in drug disposition. While targeted proteomics is an emerging technique for quantification of DMET proteins, the methodology involves significant technical challenges especially when multiple samples are analyzed in a single study over a long period of time. Therefore, it is important to thoroughly address the critical variables that could affect DMET protein quantification.

Pregnancy Increases Norbuprenorphine Clearance in Mice by Induction of Hepatic Glucuronidation.

Norbuprenorphine (NBUP) is the major active metabolite of buprenorphine (BUP) that is commonly used to treat opiate addiction during pregnancy; it possesses 25% of BUP's analgesic activity and 10 times BUP's respiratory depression effect. To optimize BUP's dosing regimen during pregnancy with better efficacy and safety, it is important to understand how pregnancy affects NBUP disposition. In this study, we examined the pharmacokinetics of NBUP in pregnant and nonpregnant mice by administering the same amount of NBUP through retro-orbital injection. We demonstrated that the systemic clearance (CL) of NBUP in pregnant mice increased ∼2.5-fold compared with nonpregnant mice. Intrinsic CL of NBUP by glucuronidation in mouse liver microsomes from pregnant mice was ∼2 times greater than that from nonpregnant mice. Targeted liquid chromatography tandem-mass spectrometry proteomics quantification revealed that hepatic Ugt1a1 and Ugt2b1 protein levels in the same amount of total liver membrane proteins were significantly increased by ∼50% in pregnant mice versus nonpregnant mice. After scaling to the whole liver with consideration of the increase in liver protein content and liver weight, we found that the amounts of Ugt1a1, Ugt1a10, Ugt2b1, and Ugt2b35 protein in the whole liver of pregnant mice were significantly increased ∼2-fold compared with nonpregnant mice. These data suggest that the increased systemic CL of NBUP in pregnant mice is likely caused by an induction of hepatic Ugt expression and activity. The data provide a basis for further mechanistic analysis of pregnancy-induced changes in the disposition of NBUP and drugs that are predominately and extensively metabolized by Ugts.

Genetic and Nongenetic Factors Associated with Protein Abundance of Flavin-Containing Monooxygenase 3 in Human Liver.

Hepatic flavin-containing mono-oxygenase 3 (FMO3) metabolizes a broad array of nucleophilic heteroatom (e.g., N or S)-containing xenobiotics (e.g., amphetamine, sulindac, benzydamine, ranitidine, tamoxifen, nicotine, and ethionamide), as well as endogenous compounds (e.g., catecholamine and trimethylamine). To predict the effect of genetic and nongenetic factors on the hepatic metabolism of FMO3 substrates, we quantified FMO3 protein abundance in human liver microsomes (HLMs; n = 445) by liquid chromatography-tandem mass chromatography proteomics. Genotyping/gene resequencing, mRNA expression, and functional activity (with benzydamine as probe substrate) of FMO3 were also evaluated. FMO3 abundance increased 2.2-fold (13.0 ± 11.4 pmol/mg protein vs. 28.0 ± 11.8 pmol/mg protein) from neonates to adults. After 6 years of age, no significant difference in FMO3 abundance was found between children and adults. Female donors exhibited modestly higher mRNA fragments per kilobase per million reads values (139.9 ± 76.9 vs. 105.1 ± 73.1; P < 0.001) and protein FMO3 abundance (26.7 ± 12.0 pmol/mg protein vs. 24.1 ± 12.1 pmol/mg protein; P < 0.05) compared with males. Six single nucleotide polymorphisms (SNPs), including rs2064074, rs28363536, rs2266782 (E158K), rs909530 (N285N), rs2266780 (E308G), and rs909531, were associated with significantly decreased protein abundance. FMO3 abundance in individuals homozygous and heterozygous for haplotype 3 (H3), representing variant alleles for all these SNPs (except rs2066534), were 50.8% (P < 0.001) and 79.5% (P < 0.01), respectively, of those with the reference homozygous haplotype (H1, representing wild-type). In summary, FMO3 protein abundance is significantly associated with age, gender, and genotype. These data are important in predicting FMO3-mediated heteroatom-oxidation of xenobiotics and endogenous biomolecules in the human liver.

Age-dependent Protein Abundance of Cytosolic Alcohol and Aldehyde Dehydrogenases in Human Liver.

Hepatic cytosolic alcohol and aldehyde dehydrogenases (ADHs and ALDHs) catalyze the biotransformation of xenobiotics (e.g., cyclophosphamide and ethanol) and vitamin A. Because age-dependent hepatic abundance of these proteins is unknown, we quantified protein expression of ADHs and ALDH1A1 in a large cohort of pediatric and adult human livers by liquid chromatography coupled with tandem mass spectrometry proteomics. Purified proteins were used as calibrators. Two to three surrogate peptides per protein were quantified in trypsin digests of liver cytosolic samples and calibrator proteins under optimal conditions of reproducibility. Neonatal levels of ADH1A, ADH1B, ADH1C, and ALDH1A1 were 3-, 8-, 146-, and 3-fold lower than the adult levels, respectively. For all proteins, the abundance steeply increased during the first year of life, which mostly reached adult levels during early childhood (age between 1 and 6 years). Only for ADH1A protein abundance in adults (age > 18 year) was ∼40% lower relative to the early childhood group. Abundances of ADHs and ALDH1A1 were not associated with sex in samples with age > 1 year compared with males. Known single nucleotide polymorphisms had no effect on the protein levels of these proteins. Quantification of ADHs and ALDH1A1 protein levels could be useful in predicting disposition and response of substrates of these enzymes in younger children.

The Promises of Quantitative Proteomics in Precision Medicine.

Precision medicine approach has a potential to ensure optimum efficacy and safety of drugs at individual patient level. Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models could play a significant role in precision medicine by predicting interindividual variability in drug disposition and response. In order to develop robust PBPK/PD models, it is imperative that the critical physiological parameters affecting drug disposition and response and their variability are precisely characterized. Currently used PBPK/PD modeling software, for example, Simcyp and Gastroplus, encompass information such as organ volumes, blood flows to organs, body fat composition, glomerular filtration rate, etc. However, the information on the interindividual variability of the majority of the proteins associated with PK and PD, for example, drug metabolizing enzymes, transporters, and receptors, are not fully incorporated into these PBPK modeling platforms. Such information is significant because the population factors such as age, genotype, disease, and gender can affect abundance or activity of these proteins. To fill this critical knowledge gap, mass spectrometry-based quantitative proteomics has emerged as an important technique to characterize interindividual variability in the protein abundance of drug metabolizing enzymes, transporters, and receptors. Integration of these quantitative proteomics data into in silico PBPK/PD modeling tools will be crucial toward precision medicine.

Age-Dependent Absolute Abundance of Hepatic Carboxylesterases (CES1 and CES2) by LC-MS/MS Proteomics: Application to PBPK Modeling of Oseltamivir In Vivo Pharmacokinetics in Infants.

The age-dependent absolute protein abundance of carboxylesterase (CES) 1 and CES2 in human liver was investigated and applied to predict infant pharmacokinetics (PK) of oseltamivir. The CES absolute protein abundance was determined by liquid chromatography-tandem mass spectrometry proteomics in human liver microsomal and cytosolic fractions prepared from tissue samples obtained from 136 pediatric donors and 35 adult donors. Two surrogate peptides per protein were selected for the quantification of CES1 and CES2 protein abundance. Purified CES1 and CES2 protein standards were used as calibrators, and the heavy labeled peptides were used as the internal standards. In hepatic microsomes, CES1 and CES2 abundance (in picomoles per milligram total protein) increased approximately 5-fold (315.2 vs. 1664.4) and approximately 3-fold (59.8 vs. 174.1) from neonates to adults, respectively. CES1 protein abundance in liver cytosol also showed age-dependent maturation. Oseltamivir carboxylase activity was correlated with protein abundance in pediatric and adult liver microsomes. The protein abundance data were then used to model in vivo PK of oseltamivir in infants using pediatric physiologically based PK modeling and incorporating the protein abundance-based ontogeny function into the existing pediatric Simcyp model. The predicted pediatric area under the curve, maximal plasma concentration, and time for maximal plasma concentration values were below 2.1-fold of the clinically observed values, respectively.

Nitric oxide synthase, calcitonin gene-related peptide and NK-1 receptor mechanisms are involved in GTN-induced neuronal activation.

BACKGROUND AND AIM: Infusion of glyceryltrinitrate (GTN), a nitric oxide (NO) donor, in awake, freely moving rats closely mimics a universally accepted human model of migraine and responds to sumatriptan treatment. Here we analyse the effect of nitric oxide synthase (NOS) and calcitonin gene-related peptide (CGRP) systems on the GTN-induced neuronal activation in this model. MATERIALS AND METHODS: The femoral vein was catheterised in rats and GTN was infused (4 µg/kg/min, for 20 minutes, intravenously). Immunohistochemistry was performed to analyse Fos, nNOS and CGRP and Western blot for measuring nNOS protein expression. The effect of olcegepant, L-nitro-arginine methyl ester (L-NAME) and neurokinin (NK)-1 receptor antagonist L-733060 were analysed on Fos activation. RESULTS: GTN-treated rats showed a significant increase of nNOS and CGRP in dura mater and CGRP in the trigeminal nucleus caudalis (TNC). Upregulation of Fos was observed in TNC four hours after the infusion. This activation was inhibited by pre-treatment with olcegepant. Pre-treatment with L-NAME and L-733060 also significantly inhibited GTN induced Fos expression. CONCLUSION: The present study indicates that blockers of CGRP, NOS and NK-1 receptors all inhibit GTN induced Fos activation. These findings also predict that pre-treatment with olcegepant may be a better option than post-treatment to study its inhibitory effect in GTN migraine models.

mRNA distribution of CGRP and its receptor components in the trigeminovascular system and other pain related structures in rat brain, and effect of intracerebroventricular administration of CGRP on Fos expression in the TNC.

Calcitonin gene-related peptide (CGRP) infusion in humans provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective against acute migraine. We hypothesized that CGRP infusion in the lateral ventricle (LV) will induce neuronal activation reflected by increase in Fos expression in the trigeminal nucleus caudalis (TNC). CGRP was infused intracerebroventricularly (i.c.v.) in freely moving rats to circumvent factors like anaesthesia, acute surgery and severe hypotension, three confounding factors for Fos expression. TNCs were isolated 2h after CGRP infusion. The level of Fos protein expression in TNC was analysed by immunohistochemistry (IHC). mRNA expression of CGRP and its receptor components in trigeminovascular and other pain processing structures in the brain was also studied. CGRP i.c.v. infusion did not induce Fos activation in the TNC. mRNA expression profile showed that CGRP and its receptor components were widely distributed in trigeminovascular and other pain processing structures. The widespread presence of CGRP receptor mRNA in the various central pain pathways suggests that CGRP might play a role in migraine pathogenesis.

A naturalistic glyceryl trinitrate infusion migraine model in the rat.

BACKGROUND AND AIM: Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose. METHODS: Awake rats were infused with GTN (4 µg/kg/min, for 20 min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively. RESULTS: A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30 min and 2 h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2 h and 4 h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4 h, demonstrating the specificity of this model for migraine. CONCLUSION: We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.

Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats.

Cisplatin is a widely used antineoplastic drug. Major drawback of cisplatin therapy is its nephrotoxicity. The objective of this study was to check the effect of tannic acid on cisplatin induced nephrotoxicity. Post-treatment of tannic acid prevents cisplatin (5mg/kg) induced nephrotoxicity and decreases poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 and hypoacetylation of histone H4. In contrast, co-treatment of tannic acid potentiates the nephrotoxicity. Comparative nephrotoxicity studies show that co-treatment of tannic acid with reduced dose of cisplatin (1.5mg/kg) developed almost similar nephrotoxicity. MALDI protein profiling of plasma samples provides indirect evidence that tannic acid co-treatment increases bioavailability of cisplatin.