Global ETD Search

301	Multiple-approaches to the identification and quantification of cytochromes P450 in human liver tissue by mass spectrometry Seibert, C., Davidson, B.R., Fuller, B.J., Patterson, Laurence H., Griffiths, W.J., Wang, Y. January 2009 (has links) No / Here we report the identification and approximate quantification of cytochrome P450 (CYP) proteins in human liver microsomes as determined by nano-LC-MS/MS with application of the exponentially modified protein abundance index (emPAI) algorithm during database searching. Protocols based on 1D-gel protein separation and 2D-LC peptide separation gave comparable results. In total, 18 CYP isoforms were unambiguously identified based on unique peptide matches. Further, we have determined the absolute quantity of two CYP enzymes (2E1 and 1A2) in human liver microsomes using stable-isotope dilution mass spectrometry, where microsomal proteins were separated by 1D-gel electrophoresis, digested with trypsin in the presence of either a CYP2E1- or 1A2-specific stable-isotope labeled tryptic peptide and analyzed by LC-MS/MS. Using multiple reaction monitoring (MRM) for the isotope-labeled tryptic peptides and their natural unlabeled analogues quantification could be performed over the range of 0.1-1.5 pmol on column. Liver microsomes from four individuals were analyzed for CYP2E1 giving values of 88-200 pmol/mg microsomal protein. The CYP1A2 content of microsomes from a further three individuals ranged from 165 to 263 pmol/mg microsomal protein. Although, in this proof-of-concept study for CYP quantification, the two CYP isoforms were quantified from different samples, there are no practical reasons to prevent multiplexing the method to allow the quantification of multiple CYP isoforms in a single sample. Amino acid sequence Chromatography Liquid Methods Cytochrome P-450 enzyme system Analysis Dilution mass spectrometry Electrophoresis Gel Two-dimensional Humans Label-free quantification LC-MS/MS 2D-LC-MS/MS Liver Metabolism Microsomes Molecular sequence data Stable isotope Tandem mass spectrometry REF 2014
302	Antitumor activity of a duocarmycin analogue rationalized to be metabolically activated by cytochrome P450 1A1 in human transitional cell carcinoma of the bladder Sutherland, Mark, Gill, Jason H., Loadman, Paul, Laye, Jonathan P., Sheldrake, Helen M., Illingworth, Nicola A., Alandas, Mohammed N., Cooper, Patricia A., Searcey, M., Pors, Klaus, Shnyder, Steven, Patterson, Laurence H. 01 October 2012 (has links) No / We identify cytochrome P450 1A1 (CYP1A1) as a target for tumor-selective drug development in bladder cancer and describe the characterization of ICT2700, designed to be metabolized from a prodrug to a potent cytotoxin selectively by CYP1A1. Elevated CYP1A1 expression was shown in human bladder cancer relative to normal human tissues. RT112 bladder cancer cells, endogenously expressing CYP1A1, were selectively chemosensitive to ICT2700, whereas EJ138 bladder cells that do not express CYP1A1 were significantly less responsive. Introduction of CYP1A1 into EJ138 cells resulted in 75-fold increased chemosensitivity to ICT2700 relative to wild-type EJ138. Negligible chemosensitivity was observed in ICT2700 in EJ138 cells expressing CYP1A2 or with exposure of EJ138 cells to CYP1B1- or CYP3A4-generated metabolites of ICT2700. Chemosensitivity to ICT2700 was also negated in EJ138-CYP1A1 cells by the CYP1 inhibitor alpha-naphthoflavone. Furthermore, ICT2700 did not induce expression of the AhR-regulated CYP1 family, indicating that constitutive CYP1A1 expression is sufficient for activation of ICT2700. Consistent with the selective activity by CYP1A1 was a time and concentration-dependent increase in gamma-H2AX protein expression, indicative of DNA damage, associated with the activation of ICT2700 in RT112 but not EJ138 cells. In mice-bearing CYP1A1-positive and negative isogenic tumors, ICT2700 administration resulted in an antitumor response only in the CYP1A1-expressing tumor model. This antitumor response was associated with detection of the CYP1A1-activated metabolite in tumors but not in the liver. Our findings support the further development of ICT2700 as a tumor-selective treatment for human bladder cancers. Animals Antineoplastic agents Biotransformation CHO cells Carcinoma Cell line Tumor Cell survival Cricetinae Cytochrome P-450 CYP1A1 Female Gene expression Humans Indoles Liver Maximum tolerated dose Mice Inbred BALB C Microsomes Pyrroles Tumor burden Urinary bladder neoplasms Xenograft model antitumor assays REF 2014 Metabolism Pharmacokinetics Pharmacology Transitional cell Drug therapy Enzymology Pathology Genetics
303	Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins Travica, S., Pors, Klaus, Loadman, Paul, Shnyder, Steven, Johansson, I., Alandas, Mohammed N., Sheldrake, Helen M., Mkrtchian, S., Patterson, Laurence H., Ingelman-Sundberg, M. January 2013 (has links) No / PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1. Animals Bystander effect Cell line Colonic neoplasms Cytochrome P-450 enzyme system Cytotoxins DNA damage Drug resistance Female Gene expression Heterocyclic compounds Humans Indoles Mice Tissue distribution Tumor burden Xenograft model antitumor assays REF 2014 Tumor 3-Ring Neoplasm Drug effects Pharmacokinetics Toxicity Metabolism Enzymology Genetics Pathology

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