44 results on '"Aryl Hydrocarbon Hydroxylases immunology"'
Search Results
2. Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry.
- Author
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Weiß F, Schnabel A, Planatscher H, van den Berg BH, Serschnitzki B, Nuessler AK, Thasler WE, Weiss TS, Reuss M, Stoll D, Templin MF, Joos TO, Marcus K, and Poetz O
- Subjects
- ATP Binding Cassette Transporter, Subfamily B immunology, ATP Binding Cassette Transporter, Subfamily B metabolism, Amino Acid Sequence, Antibodies immunology, Antibody Affinity, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Atorvastatin pharmacokinetics, Cells, Cultured, Chromatography, Liquid methods, Cytochrome P-450 CYP3A immunology, Cytochrome P-450 CYP3A metabolism, Epitopes immunology, Epitopes metabolism, Hepatocytes cytology, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacokinetics, Peptides immunology, Pravastatin pharmacokinetics, Primary Cell Culture, Sequence Homology, Amino Acid, Hepatocytes enzymology, Hepatocytes metabolism, Mass Spectrometry methods, Peptides metabolism
- Abstract
Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system.
- Published
- 2015
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3. The aryl hydrocarbon receptor is functionally upregulated early in the course of human T-cell activation.
- Author
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Prigent L, Robineau M, Jouneau S, Morzadec C, Louarn L, Vernhet L, Fardel O, and Sparfel L
- Subjects
- Active Transport, Cell Nucleus physiology, Aryl Hydrocarbon Hydroxylases biosynthesis, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Cell Nucleus genetics, Cell Nucleus metabolism, Cytochrome P-450 CYP1A1 biosynthesis, Cytochrome P-450 CYP1A1 genetics, Cytochrome P-450 CYP1A1 immunology, Cytochrome P-450 CYP1B1, Gene Knockdown Techniques, Humans, Interleukins genetics, Interleukins immunology, Interleukins metabolism, Protein Biosynthesis genetics, Protein Biosynthesis immunology, RNA, Messenger biosynthesis, RNA, Messenger genetics, RNA, Messenger immunology, Receptors, Aryl Hydrocarbon biosynthesis, Receptors, Aryl Hydrocarbon genetics, T-Lymphocytes cytology, T-Lymphocytes metabolism, Up-Regulation genetics, Interleukin-22, Cell Nucleus immunology, Lymphocyte Activation physiology, Receptors, Aryl Hydrocarbon immunology, T-Lymphocytes immunology, Up-Regulation immunology
- Abstract
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates immunosuppression caused by a variety of environmental contaminants, such as polycyclic aromatic hydrocarbons or dioxins. Recent evidence suggests that AhR plays an important role in T-cell-mediated immune responses by affecting the polarization and differentiation of activated T cells. However, the regulation of AhR expression in activated T cells remains poorly characterized. In the present study, we used purified human T cells stimulated with anti-CD3 and anti-CD28 Abs to investigate the effect of T-cell activation on AhR mRNA and protein expression. The expression of AhR mRNA increased significantly and rapidly after T-cell activation, identifying AhR as an immediate-early activation gene. AhR upregulation occurred in all of the T-cell subtypes, and is associated with its nuclear translocation and induction of the cytochromes P-450 1A1 and 1B1 mRNA expression in the absence of exogenous signals. In addition, the use of an AhR antagonist or siRNA-mediated AhR knockdown significantly inhibited IL-22 expression, suggesting that expression and functional activation of AhR is necessary for the secretion of IL-22 by activated T cells. In conclusion, our data support the idea that AhR is a major player in T-cell physiology., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2014
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4. Isoniazid hepatotoxicity: progress in understanding the immunologic component.
- Author
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James L and Roberts D
- Subjects
- Cytochrome P-450 CYP2C9, Female, Humans, Male, Aryl Hydrocarbon Hydroxylases immunology, Autoantibodies blood, Chemical and Drug Induced Liver Injury immunology, Cytochrome P-450 CYP2E1 immunology, Cytochrome P-450 CYP3A immunology, Isoniazid adverse effects
- Published
- 2014
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5. Detection of anti-isoniazid and anti-cytochrome P450 antibodies in patients with isoniazid-induced liver failure.
- Author
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Metushi IG, Sanders C, Lee WM, and Uetrecht J
- Subjects
- Adult, Aged, Antibodies blood, Antitubercular Agents adverse effects, Antitubercular Agents immunology, Chemical and Drug Induced Liver Injury epidemiology, Cytochrome P-450 CYP2C9, Female, Humans, Isoniazid immunology, Liver Failure epidemiology, Liver Failure immunology, Male, Microsomes, Liver immunology, Middle Aged, Risk Factors, Seroepidemiologic Studies, Young Adult, Aryl Hydrocarbon Hydroxylases immunology, Autoantibodies blood, Chemical and Drug Induced Liver Injury immunology, Cytochrome P-450 CYP2E1 immunology, Cytochrome P-450 CYP3A immunology, Isoniazid adverse effects
- Abstract
Unlabelled: Isoniazid (INH)-induced hepatotoxicity remains one of the most common causes of drug-induced idiosyncratic liver injury and liver failure. This form of liver injury is not believed to be immune-mediated because it is not usually associated with fever or rash, does not recur more rapidly on rechallenge, and previous studies have failed to identify anti-INH antibodies (Abs). In this study, we found Abs present in sera of 15 of 19 cases of INH-induced liver failure. Anti-INH Abs were present in 8 sera; 11 had anti-cytochrome P450 (CYP)2E1 Abs, 14 had Abs against CYP2E1 modified by INH, 14 had anti-CYP3A4 antibodies, and 10 had anti-CYP2C9 Abs. INH was found to form covalent adducts with CYP2E1, CYP3A4, and CYP2C9. None of these Abs were detected in sera from INH-treated controls without significant liver injury. The presence of a range of antidrug and autoAbs has been observed in other drug-induced liver injury that is presumed to be immune mediated., Conclusion: These data provide strong evidence that INH induces an immune response that causes INH-induced liver injury., (© 2014 by the American Association for the Study of Liver Diseases.)
- Published
- 2014
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6. Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies.
- Author
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Thiery G, Mernaugh RL, Yan H, Spraggins JM, Yang J, Parl FF, and Caprioli RM
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- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Aryl Hydrocarbon Hydroxylases chemistry, Aryl Hydrocarbon Hydroxylases immunology, Avidin chemistry, Avidin immunology, Biotin chemistry, Biotin immunology, Breast chemistry, Breast Neoplasms chemistry, Cytochrome P-450 CYP1A1 chemistry, Cytochrome P-450 CYP1A1 immunology, Cytochrome P-450 CYP1B1, Enzyme-Linked Immunosorbent Assay, Female, Histocytochemistry methods, Humans, Mice, Rats, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sensitivity and Specificity, Single-Chain Antibodies metabolism, Mass Spectrometry methods, Molecular Imaging methods, Single-Chain Antibodies chemistry
- Abstract
Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.
- Published
- 2012
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7. Immunoglobulin light chain, Blimp-1 and cytochrome P4501B1 peptides as potential vaccines for AL amyloidosis.
- Author
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Flies A, Ahmadi T, Parks AJ, Prokaeva T, Weng L, Rolfe SS, Seldin DC, and Sherr DH
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases therapeutic use, Cell Movement, Computational Biology, Cytochrome P-450 CYP1B1, Cytotoxicity, Immunologic, Disease Models, Animal, HLA-A2 Antigen genetics, HLA-A2 Antigen metabolism, Humans, Immunization, Immunoglobulin Light Chains genetics, Immunoglobulin Light Chains therapeutic use, Mice, Mice, Transgenic, Peptide Fragments genetics, Peptide Fragments immunology, Plasma Cells immunology, Positive Regulatory Domain I-Binding Factor 1, Protein Binding, T-Lymphocytes, Cytotoxic immunology, Transcription Factors genetics, Transcription Factors therapeutic use, Vaccines, Subunit genetics, Vaccines, Subunit therapeutic use, Amyloid immunology, Amyloidosis immunology, Amyloidosis therapy, Aryl Hydrocarbon Hydroxylases immunology, Immunoglobulin Light Chains immunology, Transcription Factors immunology, Vaccines, Subunit immunology
- Abstract
Amyloid light chain (AL) amyloidosis is a lethal disorder characterized by the pathologic deposition of clonal plasma cell-derived, fibrillogenic immunoglobulin light chains in vital organs. Current chemotherapeutic regimens are problematic in patients with compromised organ function and are not effective for all patients. Here, a platform of computer-based prediction and preclinical mouse modeling was used to begin development of a complementary, immunotherapeutic approach for AL amyloidosis. Three peptide/MHC I-binding algorithms identified immunogenic peptides from three AL plasma cell-associated proteins: (1) amyloidogenic λ6 light chains, (2) CYP1B1, a universal tumor antigen hyper-expressed in AL plasma cells and (3) B lymphocyte-induced maturation protein 1 (Blimp-1), a transcription factor required for plasma cell differentiation. The algorithms correctly predicted HLA-A(*)0201-binding native and heteroclitic peptides. In HLA-A2 transgenic mice, these peptides, given individually or in combination, induced potent CTL which kill peptide-loaded human lymphoma cells and/or lymphoma cells producing target protein. Blimp-1 peptide-immunized mice exhibited a reduced percentage of splenic, lymph node and bone marrow plasma cells and a decrease in the absolute number of splenic plasma cells demonstrating (1) presentation of target peptide by endogenous plasma cells and (2) appropriate CTL homing to lymphoid organs followed by killing of target plasma cells. These studies suggest that AL amyloidosis, with its relatively low tumor cell burden, may be an attractive target for peptide-based multivalent vaccines.
- Published
- 2012
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8. Immunochemical detection of cytochrome P450 enzymes in liver microsomes of 27 cynomolgus monkeys.
- Author
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Uehara S, Murayama N, Nakanishi Y, Zeldin DC, Yamazaki H, and Uno Y
- Subjects
- Animals, Anticoagulants metabolism, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Coumarins metabolism, Cytochrome P-450 CYP3A immunology, Cytochrome P-450 CYP3A metabolism, Cytochrome P-450 Enzyme System biosynthesis, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System immunology, Enzyme Assays, Female, Humans, Hydroxylation, Immunoblotting, Inactivation, Metabolic, Macaca fascicularis, Male, Microsomes, Liver metabolism, Oxazines metabolism, Sensitivity and Specificity, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver enzymology
- Abstract
The cynomolgus monkey is widely used as a primate model in preclinical studies because of its evolutionary closeness to humans. Despite their importance in drug metabolism, the content of each cytochrome P450 (P450) enzyme has not been systematically determined in cynomolgus monkey livers. In this study, liver microsomes of 27 cynomolgus monkeys were analyzed by immunoblotting using selective P450 antibodies. The specificity of each antibody was confirmed by analyzing the cross-reactivity against 19 CYP1-3 subfamily enzymes using recombinant proteins. CYP2A, CYP2B6, CYP2C9/19, CYP2C76, CYP2D, CYP2E, CYP3A4, and CYP3A5 were detected in all 27 animals. In contrast, CYP1A, CYP1D, and CYP2J were below detectable levels in all liver samples. The average content of each P450 showed that among the P450s analyzed CYP3A (3A4 and 3A5) was the most abundant (40% of total immunoquantified P450), followed by CYP2A (25%), CYP2C (14%), CYP2B6 (13%), CYP2E1 (11%), and CYP2D (3%). No apparent sex differences were found for any P450. Interanimal variations ranged from 2.6-fold (CYP3A) to 11-fold (CYP2C9/19), and most P450s (CYP2A, CYP2D, CYP2E, CYP3A4, and CYP3A5) varied 3- to 4-fold. To examine the correlations of P450 content with enzyme activities, metabolic assays were performed in 27 cynomolgus monkey livers using 7-ethoxyresorufin, coumarin, pentoxyresorufin, flurbiprofen, bufuralol, dextromethorphan, and midazolam. CYP2D and CYP3A4 contents were significantly correlated with typical reactions of human CYP2D (bufuralol 1'-hydroxylation and dextromethorphan O-deethylation) and CYP3A (midazolam 1'-hydroxylation and 4-hydroxylation). The results presented in this study provide useful information for drug metabolism studies using cynomolgus monkeys.
- Published
- 2011
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9. Transplacental transfer and metabolism of buprenorphine in preterm human placenta.
- Author
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Fokina VM, Patrikeeva SL, Zharikova OL, Nanovskaya TN, Hankins GV, and Ahmed MS
- Subjects
- Antibodies, Monoclonal, Aromatase immunology, Aromatase metabolism, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Biotransformation, Buprenorphine metabolism, Cytochrome P-450 CYP2B6, Cytochrome P-450 CYP2C8, Female, Gestational Age, Humans, In Vitro Techniques, Oxidoreductases, N-Demethylating immunology, Oxidoreductases, N-Demethylating metabolism, Perfusion, Placenta physiology, Pregnancy, Buprenorphine analogs & derivatives, Buprenorphine pharmacokinetics, Cytochrome P-450 Enzyme System metabolism, Microsomes enzymology, Placenta enzymology
- Abstract
We sought to determine whether gestational age affects the transplacental transfer and metabolism of buprenorphine (BUP). Transfer of BUP (10 ng/mL) and its [ (3)H]-isotope was determined across placentas of 30 to 34 weeks of gestation utilizing the technique of dual perfusion of placental lobule. Concentration of the drug in trophoblast tissue and in maternal and fetal circuits was determined by liquid scintillation spectrometry. Microsomes prepared from placentas of 17 to 37 weeks of gestation were divided into three groups: late second, early third, and late third trimesters. Antibodies raised against human cytochrome P450 (CYP) isoforms were utilized to identify the enzyme(s) catalyzing BUP biotransformation by preterm placental microsomes. The amount of norbuprenorphine formed was determined by liquid chromatography-mass spectrometry (LC-MS). BUP transfer across the placentas of 30 to 34 weeks of gestation was similar to those at term. CYP19 antibodies caused 60% inhibition of BUP metabolism by microsomes of late second and early third trimesters and 85% by microsomes of late third trimester. The developmental changes occurring in human placenta between 30 weeks of gestation through term do not affect the transfer of BUP across human placenta. CYP19 is the major enzyme responsible for biotransformation of BUP beginning at 17 weeks of gestation until term., (Thieme Medical Publishers.)
- Published
- 2011
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10. In vivo electroporation enhances the potency of poly-lactide co-glycolide (PLG) plasmid DNA immunization.
- Author
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Barbon CM, Baker L, Lajoie C, Ramstedt U, Hedley ML, and Luby TM
- Subjects
- Adjuvants, Immunologic pharmacology, Animals, Antigen-Presenting Cells immunology, Aryl Hydrocarbon Hydroxylases immunology, Cell Line, Tumor, Cytochrome P-450 CYP1B1, Cytokines immunology, Female, Immunity, Innate, Injections, Intramuscular, Mice, Mice, Inbred BALB C, Mice, Inbred C3H, Mice, Transgenic, Microspheres, Phagocytes immunology, Plasmids immunology, Polylactic Acid-Polyglycolic Acid Copolymer, T-Lymphocytes immunology, Electroporation, Lactic Acid pharmacology, Polyglycolic Acid pharmacology, Vaccines, DNA immunology
- Abstract
Immunization with plasmid DNA that has been encapsulated in poly lactide-co-glycolide (PLG) microparticles targets the plasmid DNA to antigen presenting cells and elicits immune responses to the encoded antigen(s). Application of a series of electrical pulses (EPT) immediately following unformulated DNA injection enhances expression of the encoded antigen and increases immune responses. The combination of using EPT before or after PLG-encapsulated plasmid DNA immunization was tested to determine if enhanced immune responses would be generated. The results show that the combination lead to both enhanced expression of antigen and more robust T cell responses, even if EPT was applied prior to immunization. The data also demonstrate that recruitment of phagocytes to the injection site was markedly enhanced by EPT, and this resulted in an increase of the antigen expression levels in these cells. Co-administration of microparticles and EPT also effected localized necrosis of muscle fibers, caused persistent Th-1-modulated cytokine production, and lead to the release of two endogenous adjuvants, uric acid and HMGB1. In all, we describe that increased immunogenicity observed with the combination of PLG-encapsulated plasmid DNA microparticle with EPT was caused by an increase in the recruitment of antigen presenting cells which mediated a more robust T cell response than observed with immunization alone., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
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11. Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite.
- Author
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Kazui M, Nishiya Y, Ishizuka T, Hagihara K, Farid NA, Okazaki O, Ikeda T, and Kurihara A
- Subjects
- Antibodies immunology, Antibodies pharmacology, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Biocatalysis, Biotransformation physiology, Cell Line, Cell Line, Tumor, Clopidogrel, Cytochrome P-450 CYP1A2 genetics, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 CYP1A2 metabolism, Cytochrome P-450 CYP1A2 Inhibitors, Cytochrome P-450 CYP2B6, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP3A genetics, Cytochrome P-450 CYP3A immunology, Cytochrome P-450 CYP3A metabolism, Cytochrome P-450 CYP3A Inhibitors, Cytochrome P-450 Enzyme System genetics, Enzyme Inhibitors pharmacology, Glutathione metabolism, Humans, Ketoconazole pharmacology, Kinetics, Mephenytoin analogs & derivatives, Mephenytoin pharmacology, Microsomes drug effects, Microsomes metabolism, Microsomes, Liver drug effects, Microsomes, Liver enzymology, NADP metabolism, Omeprazole pharmacology, Oxidation-Reduction, Oxidoreductases, N-Demethylating genetics, Oxidoreductases, N-Demethylating immunology, Oxidoreductases, N-Demethylating metabolism, Platelet Aggregation Inhibitors metabolism, Platelet Aggregation Inhibitors pharmacokinetics, Sulfaphenazole pharmacology, Theophylline analogs & derivatives, Theophylline pharmacology, Ticlopidine metabolism, Ticlopidine pharmacokinetics, Cytochrome P-450 Enzyme System metabolism, Ticlopidine analogs & derivatives
- Abstract
The aim of the current study is to identify the human cytochrome P450 (P450) isoforms involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. In the in vitro experiments using cDNA-expressed human P450 isoforms, clopidogrel was metabolized to 2-oxo-clopidogrel, the immediate precursor of its pharmacologically active metabolite. CYP1A2, CYP2B6, and CYP2C19 catalyzed this reaction. In the same system using 2-oxo-clopidogrel as the substrate, detection of the active metabolite of clopidogrel required the addition of glutathione to the system. CYP2B6, CYP2C9, CYP2C19, and CYP3A4 contributed to the production of the active metabolite. Secondly, the contribution of each P450 involved in both oxidative steps was estimated by using enzyme kinetic parameters. The contribution of CYP1A2, CYP2B6, and CYP2C19 to the formation of 2-oxo-clopidogrel was 35.8, 19.4, and 44.9%, respectively. The contribution of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 to the formation of the active metabolite was 32.9, 6.76, 20.6, and 39.8%, respectively. In the inhibition studies with antibodies and selective chemical inhibitors to P450s, the outcomes obtained by inhibition studies were consistent with the results of P450 contributions in each oxidative step. These studies showed that CYP2C19 contributed substantially to both oxidative steps required in the formation of clopidogrel active metabolite and that CYP3A4 contributed substantially to the second oxidative step. These results help explain the role of genetic polymorphism of CYP2C19 and also the effect of potent CYP3A inhibitors on the pharmacokinetics and pharmacodynamics of clopidogrel in humans and on clinical outcomes.
- Published
- 2010
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12. Consecutive low doses of cyclophosphamide preferentially target Tregs and potentiate T cell responses induced by DNA PLG microparticle immunization.
- Author
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Barbon CM, Yang M, Wands GD, Ramesh R, Slusher BS, Hedley ML, and Luby TM
- Subjects
- Animals, Antigens, Neoplasm immunology, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Cell Line, Cyclophosphamide immunology, Cytochrome P-450 CYP1B1, Drug Compounding, Epitopes immunology, Female, Humans, Immunization, Immunosuppressive Agents immunology, Mice, Mice, Inbred C3H, Spleen cytology, Spleen immunology, T-Lymphocyte Subsets immunology, T-Lymphocytes, Regulatory immunology, Cyclophosphamide pharmacology, Immunosuppressive Agents pharmacology, T-Lymphocyte Subsets drug effects, T-Lymphocytes, Regulatory drug effects, Vaccines, DNA administration & dosage, Vaccines, DNA immunology, Vaccines, DNA pharmacology
- Abstract
Cyclophosphamide in combination with immunotherapeutic approaches preferentially impinges on T(reg) activity and allows for robust generation of T cell effectors. Reduced dosages of cyclophosphamide are necessary to restrict its cytotoxic effects to the negative regulatory cell populations while sparing effector lymphocytes. We investigated cyclophosphamide dosing in combination with ZYC300, a PLG-encapsulated plasmid DNA vaccine which encodes the cytochrome P450 family member, CYP1B1, a known human tumor-associated antigen. In mice, three consecutive, low doses of cyclophosphamide comprised a superior regimen in enhancing the magnitude, diversity of epitopes, and avidity to individual epitopes of specific T cell responses when compared to regimens that used either a single low or a single high dose. Consecutive low doses of cyclophosphamide predominantly targeted T(regs) while sparing overall T lymphocyte counts. Thus, we report the synergistic activity of pharmacologic T(reg) depletion with cyclophosphamide on quantitatively and qualitatively increasing T cell responses to a known human tumor-associated antigen., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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13. Modulation of Th17 development and function by activation of the aryl hydrocarbon receptor--the role of endogenous ligands.
- Author
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Stockinger B, Veldhoen M, and Hirota K
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases metabolism, Humans, Interleukin-17 metabolism, Interleukin-23 immunology, Interleukin-23 metabolism, Interleukins metabolism, Ligands, Receptors, Aryl Hydrocarbon metabolism, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, T-Lymphocytes, Helper-Inducer metabolism, Interleukin-22, Aryl Hydrocarbon Hydroxylases immunology, Interleukin-17 immunology, Interleukins immunology, Receptors, Aryl Hydrocarbon immunology, T-Lymphocytes, Helper-Inducer immunology
- Abstract
The elucidation of the crucial differentiation factors for the new Th17 CD4 effector T-cell subset spurred an explosive growth in research publications focused on these cells and led to rapid advances in knowledge concerning their regulation and functional activities. Here we discuss recent discoveries linking the development and functional potential of Th17 cells to a transcription factor that mediates the response to exogenous and endogenous environmental signals.
- Published
- 2009
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14. Cytochrome P450 1B1 expression in rat esophageal tumorigenesis promoted by gastric and duodenal reflux.
- Author
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Devlin AH, McIlroy M, McKeen HD, Bonde P, Menezes AA, Swarbrick CJ, Robson T, Hirst DG, Campbell FC, McGuigan JA, and McKeown SR
- Subjects
- Animals, Antibody Specificity, Aryl Hydrocarbon Hydroxylases immunology, Blotting, Western, Cytochrome P-450 CYP1B1, Duodenogastric Reflux enzymology, Esophageal Neoplasms etiology, Female, Gastroesophageal Reflux enzymology, Immunohistochemistry, Mice, Mice, Knockout, RNA, Messenger genetics, Rats, Rats, Sprague-Dawley, Aryl Hydrocarbon Hydroxylases genetics, Duodenogastric Reflux complications, Esophageal Neoplasms enzymology, Gastroesophageal Reflux complications
- Abstract
Cytochrome P450 1B1 (CYP1B1) mRNA is constitutively expressed in most normal extra-hepatic tissues; however the protein is not detectable in these tissues but is expressed in a wide variety of tumors. CYP1B1 is responsible for the activation of a number of carcinogens present in tobacco smoke and food. A surgical model of rat esophageal tumorigenesis, promoted by gastric or duodenal reflux was used to determine CYP1B1 expression in premalignant esophageal tissue. Immunohistochemistry was performed using a modified amplified fluorescein tyramide protocol. CYP1B1 was not observed in normal esophageal mucosa, submucosa, or muscularis mucosa. Animals exposed to gastric reflux developed mild hyperplasia. Varying degrees of hyperplasia were observed in the duodenal reflux group. All regions of hyperplasia showed moderate or strong CYP1B1 immunoreactivity. Duodenal reflux induced a small number of premalignant changes: immunoreactivity was absent from the epithelium of squamous dysplasia (0/10), Barrett's esophagus (0/7), and majority of dysplastic Barrett's esophagus (1/4). Moderate or strong immunoreactivity was observed in the majority (7/8) of squamous cell carcinomas (SCCs) in situ. Immunoreactivity was also observed in the lamina propria and submucosa in association with inflammation, regardless of the severity of inflammation. The expression of CYP1B1 in hyperplasia, SCCs in situ, or in association with inflammation may increase the production of carcinogenic metabolites, which may promote esophageal tumorigenesis., (Copyright 2008 Wiley-Liss, Inc.)
- Published
- 2009
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15. Confirmation that cytochrome P450 2C8 (CYP2C8) plays a minor role in (S)-(+)- and (R)-(-)-ibuprofen hydroxylation in vitro.
- Author
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Chang SY, Li W, Traeger SC, Wang B, Cui D, Zhang H, Wen B, and Rodrigues AD
- Subjects
- Acetates pharmacology, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacology, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Catalysis, Cyclopropanes, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C8, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP3A genetics, Cytochrome P-450 CYP3A metabolism, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Diclofenac metabolism, Enzyme Inhibitors pharmacology, Genotype, Humans, Hydroxylation, Ibuprofen analogs & derivatives, Ketoconazole pharmacology, Kinetics, Mephenytoin analogs & derivatives, Mephenytoin pharmacology, Microsomes, Liver drug effects, Quinolines pharmacology, Recombinant Proteins metabolism, Stereoisomerism, Sulfaphenazole pharmacology, Sulfides, Tandem Mass Spectrometry, Aryl Hydrocarbon Hydroxylases metabolism, Ibuprofen metabolism, Microsomes, Liver metabolism
- Abstract
Various groups have sought to determine the impact of CYP2C8 genotype (and CYP2C8 inhibition) on the pharmacokinetics (PK) of ibuprofen (IBU) enantiomers. However, the contribution of cytochrome P450 2C8 (CYP2C8) in human liver microsomes (HLMs) has not been reported. Therefore, in vitro cytochrome P450 (P450) reaction phenotyping was conducted with selective inhibitors of cytochrome P450 2C9 (CYP2C9) and CYP2C8. In the presence of HLMs, sulfaphenazole (CYP2C9 inhibitor), and anti-CYP2C9 monoclonal antibodies (mAbs) inhibited (73-100%) the 2- and 3-hydroxylation of both IBU enantiomers (1 and 20 microM). At a higher IBU concentration (500 microM), the same inhibitors were less able to inhibit the 2-hydroxylation of (S)-(+)-IBU (32-52%) and (R)-(-)-IBU (30-64%), whereas the 3-hydroxylation of (S)-(+)-IBU and (R)-(-)-IBU was inhibited 66 to 83 and 70 to 89%, respectively. In contrast, less inhibition was observed with montelukast (CYP2C8 inhibitor, < or =35%) and anti-CYP2C8 mAbs (< or =24%) at all concentrations of IBU. When (S)-(+)-IBU and (R)-(-)-IBU (1 microM) were incubated with a panel of recombinant human P450s, only CYP2C9 formed appreciable amounts of the hydroxy metabolites. At a higher IBU enantiomer concentration (500 microM), additional P450s catalyzed 2-hydroxylation (CYP3A4, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP2B6) and 3-hydroxylation (CYP2C19). When the P450 reaction phenotype and additional clearance pathways are considered (e.g., direct glucuronidation and chiral inversion), it is concluded that CYP2C8 plays a minor role in (R)-(-)-IBU (<10%) and (S)-(+)-IBU ( approximately 13%) clearance. By extension, one would not expect CYP2C8 inhibition (and genotype) to greatly affect the pharmacokinetic profile of either enantiomer. On the other hand, CYP2C9 inhibition and genotype are expected to have an impact on the PK of (S)-(+)-IBU.
- Published
- 2008
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16. Targeting cytochrome P450 CYP1B1 with a therapeutic cancer vaccine.
- Author
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Luby TM
- Subjects
- Cytochrome P-450 CYP1B1, Humans, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases immunology, Cancer Vaccines immunology
- Abstract
The role that the immune system plays in limiting tumor formation and growth is becoming increasingly clear and passive immunotherapeutic approaches, such as the use of monoclonal antibodies, are now being successfully applied in clinical practice. Active immunization against tumors, however, has not yet been shown to have the same level of clinical efficacy. Two important reasons for this lack of efficacy have to do with the antigens being targeted, as well as the immunization approaches that have been tested. This review will highlight some of the requirements thought to be important for the successful development of an active immunization approach, with a focus on the ongoing development efforts for a novel agent targeting the cytochrome P450 family member, CYP1B1.
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- 2008
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17. [Influence repeated stress on immune responciveness and monooxigenase activity of normotensive and hypertensive rats].
- Author
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Tseĭlikman VE, Kozochkin DA, Markel' AL, Tseĭlikman OB, Sibiriak SV, Sinitskiĭ AI, Sysakov DA, and Simbitrsev AS
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Cytochrome P-450 CYP1A1 immunology, Cytochrome P-450 CYP1A1 metabolism, Cytochrome P-450 CYP2B1 immunology, Cytochrome P-450 CYP2B1 metabolism, Hypersensitivity, Delayed enzymology, Hypertension enzymology, Mixed Function Oxygenases metabolism, Rats, Rats, Wistar, Steroid Hydroxylases immunology, Steroid Hydroxylases metabolism, Stress, Physiological enzymology, Hypersensitivity, Delayed immunology, Hypertension immunology, Mixed Function Oxygenases immunology, Stress, Physiological immunology
- Abstract
Repeated stress led to antipodal directions in immune system and cytochrome P450 activities of normotensive and hypertensive rats. Enhancement of the Reaction of Delayed Hypersensitivity, suppression of cytochrome P450-mediated monooxigenase activities were observed in Wistar rats. On the contrary, in the NISAG decrease of the Reaction Delayed Hypersensitivity, elevation of cytochrome P450-mediated monooxigenase activities were observed, as comparison with Wistar rats.
- Published
- 2008
18. Use of a fluorescent substrate for the selective quantification of rat CYP3A in the liver and the intestine.
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Michaud J, Leblond FA, Naud J, Boisvert C, Desbiens K, Nicoll-Griffith DA, and Pichette V
- Subjects
- Animals, Antibodies, Blocking pharmacology, Aryl Hydrocarbon Hydroxylases analysis, Aryl Hydrocarbon Hydroxylases immunology, Cytochrome P-450 CYP3A, Fluorescence, Intestines chemistry, Liver chemistry, Male, Membrane Proteins analysis, Membrane Proteins immunology, Microsomes chemistry, Microsomes enzymology, Rats, Rats, Sprague-Dawley, Substrate Specificity, Aryl Hydrocarbon Hydroxylases metabolism, Fluorescent Dyes metabolism, Fluorobenzenes metabolism, Furans metabolism, Intestines enzymology, Liver enzymology, Membrane Proteins metabolism
- Abstract
Introduction: Quantification of cytochrome P450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P450 using Supersomes and its application in non-hepatic tissue (e.g., intestine)., Methods: Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations., Results: Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes preparations., Discussion: In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P450 in the rat liver and intestine.
- Published
- 2007
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19. CYP2A13 in human respiratory tissues and lung cancers: an immunohistochemical study with a new peptide-specific antibody.
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Zhu LR, Thomas PE, Lu G, Reuhl KR, Yang GY, Wang LD, Wang SL, Yang CS, He XY, and Hong JY
- Subjects
- Animals, Antibodies immunology, Aryl Hydrocarbon Hydroxylases immunology, Humans, Immunoblotting methods, Immunohistochemistry methods, Isoenzymes immunology, Isoenzymes metabolism, Lung Neoplasms pathology, Mice, Microsomes, Liver enzymology, Peptides immunology, Aryl Hydrocarbon Hydroxylases metabolism, Bronchi enzymology, Lung Neoplasms enzymology, Pulmonary Alveoli enzymology, Trachea enzymology
- Abstract
Human cytochrome P450 2A13 (CYP2A13) is highly efficient in the metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and another potent carcinogen, aflatoxin B1 (AFB1). Although previous studies demonstrated that CYP2A13 mRNA is predominantly expressed in human respiratory tissues, expression of CYP2A13 protein in these tissues and the involved cell types have not been determined because of the lack of CYP2A13-specific antibodies. To explore the toxicological and physiological function of CYP2A13, it is important to understand the tissue/cellular distribution of CYP2A13 protein. In this study, we generated a peptide-specific antibody against human CYP2A13 and demonstrated by immunoblot analysis that this antibody does not cross-react with heterologously expressed human CYP2A6 and mouse CYP2A5 proteins, both sharing a high degree of amino acid sequence similarity with CYP2A13. Nor does the antibody cross-react with heterologously expressed human CYP3A4, CYP2S1, or any of the cytochrome P450 enzymes present in the human liver microsomes. Using this highly specific antibody for immunohistochemical staining, we detected a high level of CYP2A13 protein expression in the epithelial cells of human bronchus and trachea, but a rare distribution in the alveolar cells. There was little expression of CYP2A13 protein in different types of lung cancers. In consideration of the high efficiency of CYP2A13 in NNK metabolic activation, our result is consistent with the reported observations that most smoking-related human lung cancers are bronchogenic and supports that CYP2A13-catalyzed in situ activation may play a critical role in human lung carcinogenesis related to NNK and AFB1 exposure.
- Published
- 2006
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20. Identification of human hepatic cytochrome P450 enzymes involved in the metabolism of 8-prenylnaringenin and isoxanthohumol from hops (Humulus lupulus L.).
- Author
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Guo J, Nikolic D, Chadwick LR, Pauli GF, and van Breemen RB
- Subjects
- Antibodies, Monoclonal, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases immunology, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 CYP1A2 Inhibitors, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C8, Dealkylation, Enzyme Inhibitors pharmacology, Flavanones isolation & purification, Humans, Hydroxylation, In Vitro Techniques, Kinetics, Liver enzymology, Microsomes, Liver metabolism, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases immunology, Omeprazole pharmacology, Quercetin pharmacology, Theophylline analogs & derivatives, Theophylline pharmacology, Xanthones isolation & purification, Aryl Hydrocarbon Hydroxylases metabolism, Cytochrome P-450 CYP1A2 metabolism, Flavanones metabolism, Humulus chemistry, Liver metabolism, Mixed Function Oxygenases metabolism, Xanthones metabolism
- Abstract
The female flowers of hops (Humulus lupulus L.) are used in the brewing of beer and are under investigation for use in dietary supplements for the management of menopausal symptoms in women. Hop extracts contain the weakly estrogenic compound isoxanthohumol (IX), proestrogenic xanthohumol, and the potent estrogen 8-prenylnaringenin (8PN). Because IX can be metabolized in the human liver to form 8PN, the specific cytochrome P450 (P450) enzymes responsible for this O-demethylation reaction were identified. In addition, the enzymes that convert IX and 8PN to their most abundant metabolites were identified because these metabolic pathways might also affect the estrogenicity of hop preparations. Specifically, the P450 enzymes that catalyze the oxidation of the prenyl side chains of IX and 8PN into trans- or cis-alcohols were investigated. Human liver microsomes and monoclonal antibodies that inhibit specific P450 enzymes were used in combination with liquid chromatography/mass spectrometry to identify the enzymes responsible for these transformations. CYP2C19 was found to catalyze the formation of both cis- and trans-alcohols of the prenyl side chain of 8PN with K(m) values of 14.8 +/- 3.2 and 16.6 +/- 4.6 microM, respectively. CYP2C8 converted 8PN regioselectively to the trans-alcohol of the prenyl group with a K(m) of 3.7 +/- 0.9 microM. Finally, CYP1A2 was found to catalyze the O-demethylation of IX to generate 8PN, with a K(m) value of 17.8 +/- 3.7 microM. These results suggest that the estrogenicity of hop constituents in vivo will depend in part on metabolic conversion that may show individual variation.
- Published
- 2006
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21. Potent blockers of the monocarboxylate transporter MCT1: novel immunomodulatory compounds.
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Guile SD, Bantick JR, Cheshire DR, Cooper ME, Davis AM, Donald DK, Evans R, Eyssade C, Ferguson DD, Hill S, Hutchinson R, Ingall AH, Kingston LP, Martin I, Martin BP, Mohammed RT, Murray C, Perry MW, Reynolds RH, Thorne PV, Wilkinson DJ, and Withnall J
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Graft vs Host Disease immunology, Herpesviridae immunology, Herpesviridae metabolism, Humans, Immunologic Factors chemistry, Monocarboxylic Acid Transporters immunology, Monocarboxylic Acid Transporters metabolism, Symporters immunology, Symporters metabolism, T-Lymphocytes immunology, T-Lymphocytes metabolism, Aryl Hydrocarbon Hydroxylases metabolism, Cytochrome P-450 Enzyme System metabolism, Herpesviridae drug effects, Immunologic Factors pharmacology, Monocarboxylic Acid Transporters antagonists & inhibitors, Symporters antagonists & inhibitors, T-Lymphocytes drug effects
- Abstract
A novel series of potent blockers of the monocarboxylate transporter, MCT1, is disclosed. From very potent but lipophilic lead compounds, systematic changes to all parts of the molecule, targeting reduction in log D, afforded compounds with significantly improved overall properties. These compounds show potent immunomodulatory activity.
- Published
- 2006
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22. Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450.
- Author
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Rehmel JL, Eckstein JA, Farid NA, Heim JB, Kasper SC, Kurihara A, Wrighton SA, and Ring BJ
- Subjects
- Antibodies, Monoclonal, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Cytochrome P-450 CYP2B6, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System genetics, Enzyme Inhibitors pharmacology, Humans, Ketoconazole pharmacology, Kinetics, Microsomes, Liver drug effects, Microsomes, Liver enzymology, Oxidoreductases, N-Demethylating genetics, Oxidoreductases, N-Demethylating immunology, Oxidoreductases, N-Demethylating metabolism, Prasugrel Hydrochloride, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins metabolism, Cytochrome P-450 Enzyme System metabolism, Piperazines metabolism, Platelet Aggregation Inhibitors metabolism, Thiophenes metabolism
- Abstract
The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.
- Published
- 2006
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23. Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib.
- Author
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Uttamsingh V, Lu C, Miwa G, and Gan LS
- Subjects
- Antibodies, Monoclonal pharmacology, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases immunology, Bortezomib, Cytochrome P-450 CYP1A2 immunology, Cytochrome P-450 CYP1A2 metabolism, Cytochrome P-450 CYP1A2 Inhibitors, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP2D6 immunology, Cytochrome P-450 CYP2D6 metabolism, Cytochrome P-450 CYP2D6 Inhibitors, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System immunology, Enzyme Inhibitors pharmacology, Humans, Kinetics, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases immunology, Recombinant Proteins metabolism, Aryl Hydrocarbon Hydroxylases metabolism, Boronic Acids metabolism, Cytochrome P-450 Enzyme System metabolism, Microsomes, Liver metabolism, Mixed Function Oxygenases metabolism, Protease Inhibitors metabolism, Pyrazines metabolism
- Abstract
VELCADE (bortezomib, PS-341), reversibly inhibits the 20S proteasome and exhibits cytotoxic and antitumor activities. Pretreatment of cancer cells with bortezomib increases the chemosensitivity of these cells, suggesting that bortezomib may be used in combination chemotherapy. The relative contributions of the five major human cytochromes P450 (P450s), 1A2, 2C9, 2C19, 2D6, and 3A4 (the focus of the present study), to the metabolism of bortezomib are an important aspect of potential drug interactions. Relative activity factor (RAF), chemical inhibition, and immunoinhibition using monoclonal antibodies were three approaches employed to determine the relative contributions of the major human P450s to the net hepatic metabolism of bortezomib. RAFs for the P450 isoform-selective substrates were determined; the ratio of the rate of metabolism of bortezomib with cDNA-expressed P450s versus rate of metabolism with human liver microsomes was normalized with respect to the RAF for each P450 isoform to determine the percentage contributions of the P450s to the net hepatic metabolism of bortezomib. CYP3A4 followed by CYP2C19 were determined to be the major contributors to the metabolism of bortezomib. Chemical inhibition and immunoinhibition confirmed that CYP3A4 and CYP2C19 were the major P450s responsible for the hepatic metabolism of bortezomib. The studies were conducted with 2 muM bortezomib, and the disappearance of bortezomib, rather than appearance of a specific metabolite, was quantified to determine the contributions of the P450s to the overall hepatic metabolism of bortezomib in humans.
- Published
- 2005
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24. Rare naturally occurring immune responses to three epitopes from the widely expressed tumour antigens hTERT and CYP1B1 in multiple myeloma patients.
- Author
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Maecker B, von Bergwelt-Baildon MS, Anderson KS, Vonderheide RH, Anderson KC, Nadler LM, and Schultze JL
- Subjects
- Adult, Aged, Aged, 80 and over, Case-Control Studies, Cell Proliferation, Cells, Cultured, Cytochrome P-450 CYP1B1, Cytotoxicity, Immunologic, Female, Humans, Interferon-gamma analysis, Male, Middle Aged, Protein Isoforms analysis, Antigens, Neoplasm immunology, Aryl Hydrocarbon Hydroxylases immunology, DNA-Binding Proteins immunology, Epitopes immunology, Multiple Myeloma immunology, T-Lymphocytes, Cytotoxic immunology, Telomerase immunology
- Abstract
The widely expressed tumour antigens hTERT and CYP1B1 are commonly expressed in multiple myeloma (MM) cells. Several trials targeting these antigens by immunotherapy have been initiated. The aim of this study was to explore whether patients with MM have an endogenous pre-existing immune response against recently identified epitopes from hTERT and CYP1B1. Peripheral blood T cells from 27 HLA-A*0201+ multiple myeloma patients at different stages of disease and 20 healthy HLA-A*0201+ donors were enriched and studied for the presence of hTERT- and CYP1B1-specific cytotoxic T cells using MHC tetramer detection and short-term ex vivo expansion. No significant expansion of tetramer-positive cells was detected in the peripheral blood of either MM patients or healthy controls when cells were stained with tetramers containing the dominant hTERT-derived epitope or two peptides derived from CYP1B1. A single ex vivo peptide stimulation led to the detection of a small population (0.3-0.5%) of hTERT-specific cells in two of 27 patients with MM. None of the patients or controls showed significant expansion of CYP1B1-specific cells after a single peptide stimulation. Thus, endogenous in vivo priming of T cells against hTERT and CYP1B1 is a rare event in MM patients. These results suggest that strategies targeting hTERT and CYP1B1 may have to utilize techniques to induce T cell responses from a naive precursor frequency.
- Published
- 2005
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25. Variability of CYP3A7 expression in human fetal liver.
- Author
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Leeder JS, Gaedigk R, Marcucci KA, Gaedigk A, Vyhlidal CA, Schindel BP, and Pearce RE
- Subjects
- Adult, Aryl Hydrocarbon Hydroxylases immunology, Biotransformation, Catalysis, Chromatography, High Pressure Liquid, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, DNA genetics, DNA isolation & purification, Dehydroepiandrosterone metabolism, Female, Genotype, Humans, Hydroxylation, Immunohistochemistry, Male, Microsomes, Liver enzymology, Pregnancy, RNA biosynthesis, RNA genetics, Reverse Transcriptase Polymerase Chain Reaction, Testosterone metabolism, Aryl Hydrocarbon Hydroxylases biosynthesis, Gene Expression Regulation, Enzymologic physiology, Liver embryology, Liver enzymology
- Abstract
Fetal liver CYP3A7 plays an important role in placental estriol synthesis during pregnancy, yet little is known concerning the extent or consequences of variability in expression. The purpose of this investigation was to characterize the variability in CYP3A7 expression using several phenotypic measures in a panel of 54 fetal livers ranging in age from 76 days to 32 weeks gestation. CYP3A7 mRNA expression was measured using quantitative polymerase chain reaction, whereas immunoreactive CYP3A7 was determined using an affinity-purified antipeptide antibody. Variability in catalytic activity was evaluated using testosterone and dehydroepiandrosterone (DHEA) as substrates. Across the entire panel, CYP3A7 was the most abundant CYP3A mRNA species present and varied 634-fold from 151 to 95,700 transcripts/ng total RNA, corrected for 18S ribosomal RNA. CYP3A4 expression was minimal based on mRNA expression (1000-fold lower than CYP3A7) and the ratio of testosterone 2alpha- (T2alphaH) to 6beta- (T6betaH) hydroxylation. T2alphaH and T6betaH were highly correlated (r(2) = 0.859), and the correlation increased (r(2) = 0.974) in livers with CYP3A5*3/*3 genotypes implying that the same enzyme (CYP3A7) generated both products. Overall, T2alphaH and DHEA16alphaH activities varied 175- and 250-fold, respectively. A subset of five samples had extremely low mRNA, protein, and catalytic activity, possibly due to pathology affecting fetal viability (anencephaly, porencephaly). In the remaining samples, T2alphaH activity varied 6.7-fold (358 +/- 142, range 97 to 643 pmol/min/mg) and DHEA16alphaH activity varied 6.2-fold (8.07 +/- 2.87, range 2.41 to 14.9 nmol/min/mg). Observed variability in CYP3A7 activity was not related to CYP3A7*2, and alternative regulatory mechanisms require further investigation.
- Published
- 2005
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26. Cytochrome P450 3A and 2B6 in the developing kidney: implications for ifosfamide nephrotoxicity.
- Author
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Aleksa K, Matsell D, Krausz K, Gelboin H, Ito S, and Koren G
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Antibodies, Monoclonal pharmacology, Antineoplastic Agents, Alkylating adverse effects, Antineoplastic Agents, Alkylating metabolism, Aryl Hydrocarbon Hydroxylases immunology, Blotting, Western, Child, Child, Preschool, Cyclophosphamide analogs & derivatives, Cyclophosphamide chemistry, Cyclophosphamide metabolism, Cytochrome P-450 CYP2B6, Cytochrome P-450 CYP3A, Fetus enzymology, Humans, Ifosfamide adverse effects, Ifosfamide analogs & derivatives, Ifosfamide chemistry, Ifosfamide metabolism, Immunohistochemistry, Infant, Kidney drug effects, Kidney metabolism, Microsomes enzymology, Microsomes metabolism, Middle Aged, Oxidoreductases, N-Demethylating immunology, Stereoisomerism, Aging metabolism, Aryl Hydrocarbon Hydroxylases metabolism, Kidney embryology, Kidney enzymology, Oxidoreductases, N-Demethylating metabolism
- Abstract
Repeated administration of agents (e.g., cancer chemotherapy) that can cause drug-induced nephrotoxicity may lead to acute or chronic renal damage. This will adversely affect the health and well-being of children, especially when the developing kidney is exposed to toxic agents that may lead to acute glomerular, tubular or combined toxicity. We have previously shown that the cancer chemotherapeutic ifosfamide (IF) causes serious renal damage substantially more in younger children (less than 3 years of age) than among older children. The mechanism of the age-related IF-induced renal damage is not known. Our major hypothesis is that renal CYP P450 expression and activity are responsible for IF metabolism to the nephrotoxic chloroacetaldehyde. Presently, the ontogeny of these catalytic enzymes in the kidney is sparsely known. The presence of CYP3A4, 3A5 and 2B6 was investigated in human fetal, pediatric and adult kidney as was the metabolism of IF (both R-IF and S-IF enantiomers) by renal microsomes to 2-dechloroethylifosfamide (2-DCEIF) and 3-dechloroethylifosfamide (3-DCEIF). Our analysis shows that CYP 3A4 and 3A5 are present as early as 8 weeks of gestation. IF is metabolized in the kidney to its two enantiomers. This metabolism can be inhibited with CYP 3A4/5 and 2B6 specific monoclonal inhibitory antibodies, whereby the CYP3A4/5 inhibitory antibody decreased the production of R-3-DCEIF by 51%, while the inhibitory CYP2B6 antibody decreased the production of S-2-DCEIF and S-3-DCEIF by 44 and 43%, respectively, in patient samples. Total renal CYP content is approximately six-fold lower than in the liver.
- Published
- 2005
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27. Unexpected association between induction of immunity to the universal tumor antigen CYP1B1 and response to next therapy.
- Author
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Gribben JG, Ryan DP, Boyajian R, Urban RG, Hedley ML, Beach K, Nealon P, Matulonis U, Campos S, Gilligan TD, Richardson PG, Marshall B, Neuberg D, and Nadler LM
- Subjects
- Aged, Antigens, Neoplasm genetics, Antigens, Neoplasm immunology, Aryl Hydrocarbon Hydroxylases genetics, Cancer Vaccines genetics, Cancer Vaccines therapeutic use, Cytochrome P-450 CYP1B1, Feasibility Studies, Female, Humans, Immunotherapy methods, Male, Middle Aged, Neoplasms therapy, Plasmids genetics, Plasmids immunology, Plasmids therapeutic use, Time Factors, Treatment Outcome, Aryl Hydrocarbon Hydroxylases immunology, Cancer Vaccines immunology, Neoplasms immunology
- Abstract
Purpose: The carcinogen activator cytochrome P450 1B1 (CYP1B1) is expressed on almost all human tumors with rare expression on normal tissues. Anti-CYP1B1-specific T cells kill CYP1B1-expressing tumors, providing the rationale to examine CYP1B1 as a target for immunotherapy., Experimental Design: ZYC300, a plasmid DNA of CYP1B1 encapsulated in biodegradable poly-DL-lactide-coglycolide microparticles, was used in a phase I clinical trial to treat 17 patients with advanced stage, progressive cancer. ZYC300 was administered i.m. at a fixed dose of 400 microg every other week for up to 12 doses., Results: Thirteen patients received six vaccinations and five received all 12 doses. No significant adverse events were observed. Six patients developed immunity to CYP1B1, three of whom developed disease stabilization. All but 1 of 11 patients who did not develop immunity to CYP1B1 progressed and did not respond to salvage therapy. Five patients who developed immunity to CYP1B1 required salvage therapy for progressive metastatic disease and showed marked response to their next treatment regimen, most of which lasted longer than 1 year., Conclusions: The association between immunity to CYP1B1 and response to next salvage therapy was not expected. Because six of the seven patients who had clinical benefit regardless of the nature of salvage therapy had developed immunity to CYP1B1, it seems highly unlikely that this occurred by chance alone. Regardless of the mechanism(s) that induced tumor regression, these findings force us to rethink how the generation of antitumor immunity might be integrated into the treatment of cancer.
- Published
- 2005
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28. Identification of a new HLA-A*0201-restricted cryptic epitope from CYP1B1.
- Author
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Maecker B, von Bergwelt-Baildon MS, Sherr DH, Nadler LM, and Schultze JL
- Subjects
- Algorithms, Antigen-Presenting Cells immunology, Antigens, Neoplasm immunology, Antigens, Neoplasm metabolism, Aryl Hydrocarbon Hydroxylases metabolism, B-Lymphocytes immunology, CD40 Antigens immunology, CD40 Antigens metabolism, Cytochrome P-450 CYP1B1, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System metabolism, Epitopes, HLA-A Antigens metabolism, HLA-A2 Antigen, Half-Life, Humans, Lymphoma metabolism, Multiple Myeloma metabolism, Neoplasm Proteins immunology, Neoplasm Proteins metabolism, Peptide Fragments metabolism, Retinoic Acid 4-Hydroxylase, T-Lymphocytes immunology, T-Lymphocytes, Cytotoxic immunology, Aryl Hydrocarbon Hydroxylases immunology, HLA-A Antigens immunology, Lymphoma immunology, Multiple Myeloma immunology, Peptide Fragments immunology
- Abstract
Cytochrome P450 1B1 (CYP1B1) was recently shown to be a candidate tumor antigen broadly expressed in solid and hematologic malignancies. Nevertheless, use of such self-antigens as targets for immune intervention can be limited because of loss of high-avidity T cells during negative selection in the thymus. Recent data suggest that targeting of cryptic epitopes may represent a way to circumvent such self-tolerance and induce efficient antitumor CTL responses. Here, we present the identification and characterization of a novel, cryptic HLA-A*0201-binding peptide from CYP1B1. The nanomer CYP246 was identified by epitope deduction using algorithms to predict HLA-A*0201-binding peptides. CYP246 is characterized by strong initial HLA-A*0201 binding but a short MHC/peptide binding half-life. Expansion of high-avidity CTL was readily possible using autologous CD40-activated B cells from normal donors and cancer patients as antigen-presenting cells, suggesting that an intact T-cell repertoire can be expanded for this epitope. Lysis of CYP1B1-expressing, HLA-A*0201+ tumor cell lines and primary tumor cells confirmed that sufficient levels of CYP246 are presented by tumor cells for effector CTL killing. These findings indicate that CYP246 is a candidate cryptic epitope for immune interventions in which tumor CYP1B1 is targeted., (Copyright 2005 Wiley-Liss, Inc)
- Published
- 2005
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29. Prediction of theophylline clearance in CCl4-treated rats using in vivo CYP1A2 and CYP3A2 contents assessed with the PKCYP test.
- Author
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Kose N, Yamamoto K, Sai Y, Isawa M, Suwa T, and Nakashima E
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Cytochrome P-450 CYP3A, Male, Membrane Proteins immunology, Microsomes, Liver enzymology, Rats, Rats, Sprague-Dawley, Recombinant Proteins metabolism, Aryl Hydrocarbon Hydroxylases metabolism, Carbon Tetrachloride Poisoning metabolism, Cytochrome P-450 CYP1A2 metabolism, Membrane Proteins metabolism, Midazolam pharmacokinetics, Theophylline pharmacokinetics
- Abstract
We previously established a method to predict the drug metabolism capacity of injured liver based on pharmacokinetic estimation of the amount of cytochrome P450 (CYP) in vivo (PKCYP test), by introducing the apparent liver-to-blood free concentration gradient in vivo (qg) as a parameter. Here we show that the amount of CYP3A2 in CCl(4)-treated rats can be estimated appropriately by applying the PKCYP test using midazolam (MDZ) as a probe, assuming that the qg value in control rats does not change. We applied the results to predict the clearance of theophylline as a model drug with a physiologically based pharmacokinetic model. Male Sprague-Dawley rats were pretreated with CCl4, and the amount of CYP (A-CYP(vivo)) was quantified by Western blotting. The qg value of MDZ was determined in control rats and used to estimate the amounts of CYP3A2 in CCl4-treated rats; the result agreed well with the observed values. The qg value of CYP3A2 estimated with MDZ as a probe was used together with our previously reported value for CYP1A2 (theophylline metabolism in the liver is known to be almost entirely mediated by CYP3A2 and CYP1A2) to predict the total body clearance (CL(tot)) of theophylline in CCl4-treated rats. The predicted CL(tot) was about one-third of the observed value, which was considered acceptable. The time-course of theophylline concentration in serum simulated with a physiologically-based pharmacokinetic model agreed well with the observed values. Thus, the PKCYP test using MDZ as a probe can be used to predict the amount of CYP3A2 and the CL(tot) of theophylline in CCl4-treated rats.
- Published
- 2005
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30. Conversion of the HIV protease inhibitor nelfinavir to a bioactive metabolite by human liver CYP2C19.
- Author
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Hirani VN, Raucy JL, and Lasker JM
- Subjects
- Antibodies, Monoclonal pharmacology, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases immunology, Biotransformation, Cytochrome P-450 CYP2C19, Enzyme Inhibitors pharmacology, Humans, Hydroxylation, In Vitro Techniques, Kinetics, Mixed Function Oxygenases antagonists & inhibitors, Mixed Function Oxygenases immunology, Nelfinavir metabolism, Omeprazole pharmacology, Aryl Hydrocarbon Hydroxylases metabolism, HIV Protease Inhibitors pharmacokinetics, Microsomes, Liver metabolism, Mixed Function Oxygenases metabolism, Nelfinavir analogs & derivatives, Nelfinavir pharmacokinetics
- Abstract
Antiretroviral therapy for human immunodeficiency virus (HIV) infection includes treatment with both reverse transcriptase inhibitors and protease inhibitors, which markedly suppress viral replication and circulating HIV RNA levels. Cytochrome P450 (P450) enzymes in human liver, chiefly CYP3A4, play a pivotal role in protease inhibitor biotransformation, converting these agents to largely inactive metabolites. However, the protease inhibitor nelfinavir (Viracept) is metabolized mainly to nelfinavir hydroxy-t-butylamide (M8), which exhibits potent antiviral activity, and to other minor products (termed M1 and M3) that are inactive. Since indirect evidence suggests that CYP2C19 underlies M8 formation, we examined the role of this inducible, polymorphic P450 enzyme in nelfinavir t-butylamide hydroxylation by human liver. Rates of microsomal M8 formation were 50.6 +/- 28.3 pmol of product formed/min/nmol P450 (n = 5 subjects), whereas kinetic analysis of the reaction revealed a KM of 21.6 microM and a Vmax of 24.6 pmol/min/nmol P450. In reconstituted systems, CYP2C19 catalyzed nelfinavir t-butylamide hydroxylation at a turnover rate of 2.2 min(-1), whereas CYP2C9, CYP2C8, and CYP3A4 were inactive toward nelfinavir. Polyclonal anti-CYP2C9 (cross-reactive with CYP2C19) and monoclonal anti-CYP2C19 completely inhibited microsomal M8 production, whereas monoclonal CYP2C9 and polyclonal CYP3A4 antibodies were without effect. Similarly, the CYP2C19 substrate omeprazole strongly inhibited (75%) hepatic nelfinavir t-butylamide hydroxylation at a concentration of only 12.5 microM. Our study shows that CYP2C19 underlies formation in human liver of M8, a bioactive nelfinavir metabolite. The inducibility of CYP2C19 by agents (e.g., rifampicin) often taken concurrently with nelfinavir, together with this P450's known polymorphic nature, may thus be important determinants of nelfinavir's antiviral potency.
- Published
- 2004
- Full Text
- View/download PDF
31. High levels of autoantibodies against drug-metabolizing enzymes in SLA/LP-positive AIH-1 sera.
- Author
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Shinoda M, Tanaka Y, Kuno T, Matsufuji T, Matsufuji S, Murakami Y, and Mizutani T
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 immunology, Adult, Aryl Hydrocarbon Hydroxylases immunology, Cytochrome P-450 CYP2A6, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System immunology, Enzyme-Linked Immunosorbent Assay, Female, Glucuronosyltransferase immunology, Humans, Male, Microsomes, Liver immunology, Middle Aged, Mixed Function Oxygenases immunology, Autoantibodies blood, Autoantigens immunology, Hepatitis, Autoimmune immunology
- Abstract
Autoimmune hepatitis type 1 (AIH-1) is characterized by the detection of smooth muscle autoantibodies, antinuclear antibodies and antineutrophil cytoplasmic autoantibodies, and AIH-2 is characterized by the presence of autoantibodies against LKM, which contain drug-metabolizing enzymes. In this study, we measured the levels of drug-metabolizing enzymes in AIH-1 patients (ANA-positive). We exhaustively investigated the level of autoantibodies against major CYPs and UDP-glucuronosyltransferases of typical phase II drug-metabolizing enzymes, a transporter (MDR1), and NADPH-cytochrome P450 reductase in 4 patients with AIH-1 and 6 controls, as a case report. Two (Patients 3 and 4) of the AIH patients exhibited high levels of autoantibodies, while two (Patients 1 and 2) of the patients and the controls did not. The levels of autoantibodies against CYP2C19, CYP2D6, CYP2E1, UGT1A6 and human liver microsomes in Patients 3 and 4 sera were over 2(3) times the levels in Patient 1, Patient 2 and the control sera. Meanwhile, the levels of autoantibodies against CYP1A2, CYP2A6, CYP2C9, UGT2B7, MDR1 and NADPH-cytochrome P450 reductase were 2-2(2) higher in Patients 3 and 4 than in the other subjects. We found that the pattern of elevation in the Patient 3 serum was not parallel with that in Patient 4. Thus, we found high levels of autoantibodies against drug-metabolizing enzymes in AIH-1 patients.
- Published
- 2004
- Full Text
- View/download PDF
32. Strain differences in diazepam metabolism at its three metabolic sites in sprague-dawley, brown norway, dark agouti, and wistar strain rats.
- Author
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Saito K, Sakai N, Kim HS, Ishizuka M, Kazusaka A, and Fujita S
- Subjects
- Alcohol Oxidoreductases, Animals, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Blotting, Western methods, Chromatography, High Pressure Liquid methods, Cytochrome P-450 CYP3A, Cytochrome P450 Family 2, Diazepam antagonists & inhibitors, Diazepam pharmacology, Hydroxylation drug effects, Immune Sera immunology, Immune Sera metabolism, Kinetics, Male, Membrane Proteins genetics, Membrane Proteins immunology, Membrane Proteins metabolism, Microsomes, Liver drug effects, Microsomes, Liver enzymology, NADP metabolism, Rats, Rats, Inbred BN, Rats, Inbred Strains, Rats, Mutant Strains, Rats, Sprague-Dawley, Rats, Wistar, Steroid 16-alpha-Hydroxylase genetics, Steroid 16-alpha-Hydroxylase immunology, Steroid 16-alpha-Hydroxylase metabolism, Temazepam metabolism, Diazepam metabolism, Polymorphism, Genetic genetics, Species Specificity, Temazepam analogs & derivatives
- Abstract
Knowledge of strain differences in drug metabolism is important for the selection of animals for pharmacokinetic, pharmacodynamic, and toxicological studies. Hepatic microsomes from Sprague-Dawley (SD) and Brown Norway (BN) rats had 300-fold higher diazepam p-hydroxylation activity than Dark Agouti (DA) and Wistar (W) rats at a low diazepam concentration (3 microM). Kinetic studies indicated that diazepam p-hydroxylation in SD and BN rats proceeded with lower K(m) and higher V(max) values than it did in DA and W rats. However, the expression levels of cytochrome P450 CYP2D1, the reported enzyme for diazepam p-hydroxylation, did not cosegregate with the activity. These results suggest the presence of a new high-affinity diazepam p-hydroxylation enzyme other than CYP2D1 in SD and BN rats. DA rats showed 3- and 2-fold higher diazepam 3-hydroxylation and N-desmethylation activities, respectively, than the other rat strains. In agreement with this, DA rat liver microsomes had a higher expression of CYP3A2, which is responsible for diazepam 3-hydroxylation and partly responsible for N-desmethylation. Values of CL(int) (V(max)/K(m)) indicated that p-hydroxy-diazepam is the major metabolite in SD and BN rats, whereas 3-hydroxy-diazepam is the major metabolite in DA and W rats. The sum of the CL(int) in each strain was in the order of DA > SD = BN >> W. Strain differences in the pharmacodynamics of diazepam between SD and DA rats may be due to these differences in diazepam metabolism. We found that both the rate of elimination of diazepam and the major metabolic pathways in diazepam metabolism differed among the different rat strains due to polymorphic expression of the two enzymes involved in diazepam metabolism.
- Published
- 2004
33. Autoimmune hepatitis: evolving concepts.
- Author
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Diamantis I and Boumpas DT
- Subjects
- Ammonia-Lyases immunology, Argininosuccinate Lyase immunology, Aryl Hydrocarbon Hydroxylases immunology, Asialoglycoprotein Receptor immunology, Autoantibodies immunology, Hepatitis, Autoimmune etiology, Hepatitis, Autoimmune genetics, Hepatitis, Autoimmune pathology, Humans, Liver pathology, RNA, Transfer immunology, Antibody-Dependent Cell Cytotoxicity immunology, Autoantigens immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Hepatitis, Autoimmune immunology, Liver immunology
- Abstract
The liver is continuously exposed to a large antigenic load that includes pathogens, toxins, tumor cells and dietary antigens. A loss of tolerance against its own antigens may result in autoimmune hepatitis (AIH). The current paradigm holds that the disease is the result of self-perpetuating autoimmune process triggered by yet unknown factors (infections, chemicals, drugs) in a genetically susceptible host. To date, several putative hepatocellular surface antigens have been identified: P450-IID6 (recognized by the anti-LKM-1 autoantibodies) a membrane bound asialoglycoprotein receptor (a liver-specific membrane protein), a cytosolic UGA-suppressor tRNA associated protein (recognized by anti-SMA and anti-LP antibodies) and argininosuccinate lysate and formiminotransferase cyclodeaminase (recognized by ant-LC1 antibodies). In contrast to other chronic hepatitides patients with AIH display significant T cell hypereactivity to autologous liver antigens. Tissue injury seems to be mediated by CD4+ or CD8+ T cells and/or by antibody-dependent cell mediated cytotoxicity.
- Published
- 2004
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- View/download PDF
34. Effect of gemfibrozil on the metabolism of pitavastatin--determining the best animal model for human CYP and UGT activities.
- Author
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Fujino H, Saito T, Tsunenari Y, and Kojima J
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Dogs, Glucuronosyltransferase metabolism, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors metabolism, Immune Sera immunology, Lactones metabolism, Macaca fascicularis, Male, Metabolic Clearance Rate, Microsomes, Liver drug effects, Microsomes, Liver metabolism, Models, Animal, Quinolines antagonists & inhibitors, Rats, Rats, Sprague-Dawley, Enzyme Inhibitors metabolism, Gemfibrozil pharmacology, Hypolipidemic Agents pharmacology, Quinolines metabolism
- Abstract
A series of studies was conducted to determine the best animal model for human CYP and UGT activities. The investigation focused primarily on the interactions occurring in the CYP- or UGT-mediated metabolism of pitavastatin, and involved in vitro and in vivo experiments. We found that the best animal models for human CYP-mediated hydroxylation and UGT-mediated lactonization of pitavastatin were rats and dogs, respectively. In addition, a large difference in the metabolic properties of pitavastatin was found between monkeys and humans. In the presence of gemfibrozil, the CYP- or UGT-mediated metabolism of pitavastatin was inhibited in vitro. However, gemfibrozil treatment had no inhibitory effect on the AUC of pitavastatin and its lactone form in rats and dogs. We conclude that the plasma level of pitavastatin would not be increased by co-administration of gemfibrozil in humans.
- Published
- 2004
- Full Text
- View/download PDF
35. Cytochrome P450 2A6: a new hepatic autoantigen in patients with chronic hepatitis C virus infection.
- Author
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Dalekos GN, Obermayer-Straub P, Bartels M, Maeda T, Kayser A, Braun S, Loges S, Schmidt E, Gershwin ME, and Manns MP
- Subjects
- Adolescent, Adult, Autoantibodies analysis, Autoimmune Diseases immunology, Case-Control Studies, Child, Chronic Disease, Cytochrome P-450 CYP2A6, Female, Flaviviridae Infections immunology, Hepatitis, Viral, Human immunology, Humans, Liver Diseases immunology, Male, Middle Aged, Rheumatic Diseases immunology, Aryl Hydrocarbon Hydroxylases immunology, Autoantigens immunology, Hepatitis C, Chronic immunology, Mixed Function Oxygenases immunology
- Abstract
Background/aims: Cytochromes P4502A6 (CYP2A6) and P4501A2 (CYP1A2) were described as hepatic autoantigens in the autoimmune polyglandular syndrome type-1 (APS-1). We evaluated the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic diseases in the absence of APS-1., Methods: A radioligand assay (RLA) based on immunoprecipitation of [(35)S]-methionine-labeled CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects with chronic viral hepatitis (n=185), autoimmune liver diseases (n=181), autoimmune rheumatic diseases (ARD, n=31) and healthy (n=33) were tested., Results: Seven out of 366 patients with liver diseases were anti-CYP2A6 positive. Neither healthy nor ARD patients showed anti-CYP2A6. One out of 181 patients with autoimmune liver diseases tested anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6 (P<0.05) was detected with six out of seven patients positive in the viral hepatitis group. The latter were infected by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive sera were positive for anti-LKM-1 by immunofluorescence and for anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2., Conclusions: For the first time CYP2A6 is reported as a hepatic autoantigen in patients with viral hepatitis caused by flaviviruses and in particular in HCV/anti-LKM-1 positive patients. Multicenter studies are needed in order to investigate the clinical importance of this novel finding. This study further supports that anti-CYP2A6 in the absence of flavivirus is rather limited to APS-1.
- Published
- 2003
- Full Text
- View/download PDF
36. The shared tumor-associated antigen cytochrome P450 1B1 is recognized by specific cytotoxic T cells.
- Author
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Maecker B, Sherr DH, Vonderheide RH, von Bergwelt-Baildon MS, Hirano N, Anderson KS, Xia Z, Butler MO, Wucherpfennig KW, O'Hara C, Cole G, Kwak SS, Ramstedt U, Tomlinson AJ, Chicz RM, Nadler LM, and Schultze JL
- Subjects
- Animals, Blood Cells, Cell Line, Tumor, Cytochrome P-450 CYP1B1, Cytotoxicity, Immunologic, Epitopes, T-Lymphocyte immunology, HLA-A Antigens, HLA-A2 Antigen genetics, HLA-A2 Antigen immunology, Humans, Mice, Mice, Transgenic, Neoplasms blood, Neoplasms pathology, Peptides immunology, Antigens, Neoplasm immunology, Aryl Hydrocarbon Hydroxylases immunology, Neoplasms immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Cytochrome P450 1B1 (CYP1B1), a drug-metabolizing extrahepatic enzyme, was recently shown to be overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for anticancer therapy, particularly cancer immunotherapeutics. We identified HLA-A*0201-binding peptides and a naturally processed and presented T-cell epitope capable of inducing CYP1B1-specific cytotoxic T lymphocytes (CTLs) in HLA-A2 transgenic mice. Furthermore, the induction of CYP1B1-specific T cells was demonstrated in healthy donors and cancer patients. These T cells efficiently lysed target cells pulsed with the cognate peptide. More important, HLA-A2-matched tumor cell lines and primary malignant cells were also recognized by CYP1B1-specific CTLs. These findings form the basis of a phase 1 clinical trial exploring a DNA-based vector encoding CYP1B1 for widely applicable cancer immunotherapy conducted at the Dana-Farber Cancer Institute.
- Published
- 2003
- Full Text
- View/download PDF
37. Cytochrome P450 2A6 meets P450 2D6: an enigma of viral infections and autoimmunity.
- Author
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Bogdanos DP and McFarlane IG
- Subjects
- Cytochrome P-450 CYP2A6, Humans, Aryl Hydrocarbon Hydroxylases immunology, Autoimmune Diseases immunology, Cytochrome P-450 CYP2D6 immunology, Hepatitis C immunology, Mixed Function Oxygenases immunology
- Published
- 2003
- Full Text
- View/download PDF
38. Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine.
- Author
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Malaplate-Armand C, Ferrari L, Masson C, Siest G, and Batt AM
- Subjects
- Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Astrocytes drug effects, Astrocytes immunology, Astrocytoma, Cytochrome P-450 CYP1B1, Cytochrome P-450 CYP2D6 genetics, Cytochrome P-450 CYP2D6 immunology, Cytochrome P-450 CYP2D6 metabolism, Drug Interactions, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic immunology, Humans, Interleukin-1 antagonists & inhibitors, Isoenzymes genetics, Isoenzymes immunology, Isoenzymes metabolism, Microsomes metabolism, Oxidative Stress drug effects, RNA, Messenger biosynthesis, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Tumor Cells, Cultured, Up-Regulation immunology, Acetylcysteine pharmacology, Aryl Hydrocarbon Hydroxylases metabolism, Astrocytes enzymology, Interleukin-1 toxicity, Oxidative Stress immunology
- Abstract
The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect., (Copyright 2002 Elsevier Science Ireland Ltd.)
- Published
- 2003
- Full Text
- View/download PDF
39. Different contribution of CYP2C19 in the in vitro metabolism of three proton pump inhibitors.
- Author
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Kita T, Sakaeda T, Baba T, Aoyama N, Kakumoto M, Kurimoto Y, Kawahara Y, Okamura N, Kirita S, Kasuga M, and Okumura K
- Subjects
- 2-Pyridinylmethylsulfinylbenzimidazoles, Adult, Analysis of Variance, Antibodies pharmacology, Aryl Hydrocarbon Hydroxylases immunology, Chromatography, High Pressure Liquid instrumentation, Chromatography, High Pressure Liquid methods, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System immunology, Cytochrome P-450 Enzyme System metabolism, Dose-Response Relationship, Drug, Drug Interactions, Enzyme Inhibitors metabolism, Female, Humans, In Vitro Techniques, Lansoprazole, Male, Middle Aged, Mixed Function Oxygenases immunology, Proton-Translocating ATPases antagonists & inhibitors, Rabeprazole, Aryl Hydrocarbon Hydroxylases metabolism, Benzimidazoles metabolism, Mixed Function Oxygenases metabolism, Omeprazole analogs & derivatives, Omeprazole metabolism, Proton Pump Inhibitors
- Abstract
A series of clinical studies on the cytochrome P450 2C19 (CYP2C19) genotype and the pharmacokinetics and pharmacodynamics of three proton pump inhibitors (PPIs), omeprazole, lansoprazole and rabeprazole, have been conducted to establish the individualized pharmacotherapy based on the CYP2C19 genotyping, and in the present study, the CYP2C19 genotype-dependency was more pronounced for omeprazole than the other two. Herein, to validate further the difference among 3 PPIs in CYP2C19 genotype-dependency on the phenotype, a comparative in vitro study was conducted using the human liver microsomes and newly developed anti-human CYP antibodies. The residual concentrations of omeprazole and lansoprazole in 5 lots of human liver microsomes were dependent on the CYP2C19 activities, however, for rabeprazole, there was no correlation. The hydroxylation of omeprazole was more inhibited by anti-CYP2C19 antibody than lansoprazole, whereas anti-CYP3A4 antibody showed similar inhibition. In conclusion, the relative contribution of CYP2C19 on total metabolism of 3 PPIs elucidated herein coincided with the CYP2C19 genotype-dependent pharmacokinetics.
- Published
- 2003
- Full Text
- View/download PDF
40. Rat and human liver cytochrome P-450 isoform metabolism of ecteinascidin 743 does not predict gender-dependent toxicity in humans.
- Author
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Reid JM, Kuffel MJ, Ruben SL, Morales JJ, Rinehart KL, Squillace DP, and Ames MM
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Aryl Hydrocarbon Hydroxylases genetics, Aryl Hydrocarbon Hydroxylases immunology, Bile chemistry, Cytochrome P-450 CYP1A1 metabolism, Cytochrome P-450 CYP1A2 metabolism, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System metabolism, DNA, Complementary genetics, Dioxoles adverse effects, Dioxoles chemistry, Dioxoles metabolism, Enzyme Inhibitors pharmacology, Female, Half-Life, Humans, Immune Sera, Isoquinolines adverse effects, Isoquinolines chemistry, Isoquinolines metabolism, Male, Marine Toxins adverse effects, Marine Toxins chemistry, Marine Toxins metabolism, Molecular Structure, NADP pharmacology, Oxidoreductases, N-Demethylating antagonists & inhibitors, Oxidoreductases, N-Demethylating genetics, Oxidoreductases, N-Demethylating immunology, Protein Isoforms antagonists & inhibitors, Protein Isoforms metabolism, Rats, Rats, Inbred F344, Recombinant Fusion Proteins metabolism, Steroid Hydroxylases genetics, Steroid Hydroxylases metabolism, Substrate Specificity, Tetrahydroisoquinolines, Trabectedin, Urochordata chemistry, Aryl Hydrocarbon Hydroxylases metabolism, Dioxoles pharmacokinetics, Isoquinolines pharmacokinetics, Marine Toxins pharmacokinetics, Microsomes, Liver enzymology, Oxidoreductases, N-Demethylating metabolism, Sex Characteristics
- Abstract
Ecteinascidin 743 (ET743, NSC648766) is a marine natural product with potent in vivo activity in human xenograft models. Hepatotoxicity was the most prominent toxicity in preclinical studies and was greater in female rats than in male rats. To assess the potential implications for human toxicities, the in vitro metabolism of ET743 was characterized using rat and human preparations. NADPH-dependent ET743 metabolism was greater with male rat liver microsomal preparations than with preparations from female rats and was induced by pretreatment of rats with phenobarbital and dexamethasone but not by pretreatment with 3-methylcholanthrene. Rat and human microsomal metabolism of ET743 was reduced in the presence of chemical CYP3A inhibitors or antirat CYP3A2 antiserum and to a much lesser extent by CYP2E, CYP2C, and CYP2A inhibitors. In human liver panel studies, ET743 disappearance was highly correlated with CYP3A activities and to a lesser extent with CYP2C activities. ET743 was metabolized by a number of cDNA-expressed rat P-450 isoforms, including male-predominant CYP2A2 and CYP3A2. ET743 was metabolized by cDNA-expressed human CYP3A4 and to a much lesser extent by CYP2C9, CYP2D6, and CYP2E1 preparations. Three oxidative metabolites were detected in cDNA-expressed isoform incubations, including the N-demethylated metabolite ET729 and two additional products characterized by laser capture-mass spectrometry analyses. The plasma pharmacokinetics and biliary excretion of ET743 were characterized in rats. There were no gender-dependent differences in half-life or total body clearance values. Although very modest, the biliary excretion of ET743 in male rats (0.48%) was greater than in female rats (0.28%). In contrast, the biliary excretion of the cytotoxic N-demethylated metabolite ET729 was 5-fold greater in the female rat (1.05% of dose) than in the male rat (0.19% of dose). Biliary excretion of ET729 may contribute to the hepatic toxicity in rats. These data are consistent with a major role for CYP3A isoforms in ET743 rat and human metabolism. Although there are conflicting data in the literature, expression of CYP3A isoforms in human tissues and elimination of CYP3A substrates have not been shown to vary substantially by gender. There are no indications that the other CYP isoforms implicated in ET743 metabolism are expressed differently in males and females. Thus, although it is not possible to rule out gender differences in ET743 human toxicities, our data do not predict major gender-dependent differences in the toxicity of ET743 based on metabolism.
- Published
- 2002
41. Universal tumor antigens as targets for immunotherapy.
- Author
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Gordan JD and Vonderheide RH
- Subjects
- Antigens, Neoplasm classification, Antigens, Neoplasm genetics, Antigens, Neoplasm metabolism, Aryl Hydrocarbon Hydroxylases immunology, Aryl Hydrocarbon Hydroxylases metabolism, Clinical Trials as Topic, Computational Biology, Cytochrome P-450 CYP1B1, DNA-Binding Proteins, Epitopes immunology, Humans, Inhibitor of Apoptosis Proteins, Microtubule-Associated Proteins immunology, Microtubule-Associated Proteins metabolism, Neoplasm Proteins, Proto-Oncogene Proteins immunology, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-mdm2, Survivin, T-Lymphocytes immunology, T-Lymphocytes metabolism, Telomerase immunology, Telomerase metabolism, Antigens, Neoplasm immunology, Immunotherapy methods, Neoplasms therapy, Nuclear Proteins
- Abstract
Background: Clinically successful Ag-specific cancer immunotherapy depends on the identification of tumor-rejection Ags. Historically, tumor Ags have been identified by analyzing cancer patients' own T-cell or Ab responses., Methods: The unveiling of the human genome and optimized immunological analytical tools, particularly 'reverse immunology', have made it possible to screen any given protein for immunogenic epitopes. These advances enable the immunological characterization of universal tumor-associated gene products that mediate critical functions for tumor growth and development., Results: Four examples of candidate universal tumor Ags reviewed here include the telomerase reverse transcriptase (hTERT), the inhibitor of apoptosis survivin, the p53-interacting protein MDM2, and the cytochrome P450 isoform 1B1--each at various levels of preclinical and clinical development., Discussion: The cardinal feature of universal TAA is that they are expressed in (nearly) all tumors and in no normal tissues. They are directly involved in the malignant phenotype of the tumor. Certain peptides derived from such Ags are expressed on the tumor-cell surface, as evidenced by Ag-specific, MHC-restricted T-cell anti-tumor reactivity in vitro. It is hoped that these features imply a pre-existing, high-affinity T-cell pool that can be activated in vivo in patients, without immunoselection of variant tumor cells no longer expressing the Ag of choice.
- Published
- 2002
- Full Text
- View/download PDF
42. Purification and properties of a new beta-naphthoflavone inducible cytochrome P-450, aryl hydrocarbon hydroxylase from rat kidney.
- Author
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Ohgiya N, Yokota H, Mitsuru, Takahashi, Komoro S, and Yuasa A
- Subjects
- Amino Acid Sequence, Animals, Aryl Hydrocarbon Hydroxylases biosynthesis, Aryl Hydrocarbon Hydroxylases immunology, Carcinogens metabolism, Enzyme Induction, Male, Molecular Sequence Data, Peptide Mapping, Rats, Rats, Wistar, beta-Naphthoflavone, Aryl Hydrocarbon Hydroxylases isolation & purification, Benzoflavones pharmacology, Kidney enzymology
- Abstract
In rat kidney, beta-naphthoflavone induced 53 kDa and 55 kDa proteins, which were both recognized by the antibodies against rat liver cytochrome P-450 1A1 (55kDa). The major inducible 53 kDa protein was purified from the beta naphthoflavone-treated rat kidney and shown to be a new cytochrome P-450 having a high aryl hydrocarbon hydroxylase activity. Purified cytochrome P-450, named P-450KAh, was homogeneous on SDS-polyacrylamide gel electrophoresis, and the apparent molecular weight was estimated to be 53 kDa. The absorption spectra of the oxidized form of P-450KAh showed a Soret peak at 416 nm, a characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 446 nm. In the reconstituted system, purified P-450KAh showed high catalytic activity for benzo[a]pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. P-450KAh could activate genotoxicities of not only B[a]P, but also 2-acetylaminofluorene and aflatoxin B1 on the umu test. These catalytic properties of P-450KAh were almost the same as those of P-4501A1, a major P-450 form having arylhydrocarbon hydroxylase in liver microsomes of 3-methylcholanthrene-treated rats, and P-450KAh could not be distinguished from P-4501A1 even by immunochemical analysis. However, the electrophoretic peptide patterns after alpha-chymotrypsin or trypsin treatment of P-450KAh were different from those of P-4501A1, and the NH2-terminal 11 amino acid sequence of the P-450 was also different from that of P-4501A1 and any other P-450s of rat.
- Published
- 1996
- Full Text
- View/download PDF
43. Quantitation of hepatic cytochrome P1-450 mRNA with the use of a cloned DNA probe. Effects of various P-450 inducers in C57BL/6N and DBA/2N mice.
- Author
-
Tukey RH, Negishi M, and Nebert DW
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases immunology, Cloning, Molecular, Cytochrome P-450 Enzyme System biosynthesis, Cytochrome P-450 Enzyme System immunology, Enzyme Induction drug effects, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, Microsomes, Liver enzymology, Nucleic Acid Hybridization, Cytochrome P-450 Enzyme System metabolism, DNA, Liver enzymology, RNA, Messenger metabolism
- Published
- 1982
44. Aryl hydrocarbon hydroxylase is inhibited by antibody to rat liver cytochrome P-450.
- Author
-
Robie-Suh K, Robinson R, Gelboin HV, and Guengerich FP
- Subjects
- Animals, Antigen-Antibody Reactions, Aryl Hydrocarbon Hydroxylases immunology, Benz(a)Anthracenes pharmacology, Enzyme Induction drug effects, Humans, Methylcholanthrene pharmacology, Pentobarbital pharmacology, Rats, Aryl Hydrocarbon Hydroxylases antagonists & inhibitors, Cytochrome P-450 Enzyme System immunology, Lymphocytes enzymology, Monocytes enzymology
- Abstract
Antibody to the major purified cytochrome P-450 induced by 3-methylcholanthrene in rat liver strongly inhibits aryl hydrocarbon hydroxylase activity of uninduced and benz[a]anthracene-induced human monocytes and lymphocytes. Antibody to the cytochrome P-450 induced by phenobarbital has relatively little or no effect on the aryl hydrocarbon hydroxylase activity of the same human cells.
- Published
- 1980
- Full Text
- View/download PDF
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