ABSTRACT
Recent studies indicate that the Tg2576 transgenic mouse model of Alzheimer's disease [tg(hAPP)] demonstrates disturbances in plasma glucose and neuroendocrine function reminiscent of Alzheimer's disease (AD). Alterations in any one of these systems can have a profound effect on hepatic cytochrome P450 (CYP) expression. Additionally, the recent discovery that amyloid beta 1-42 can induce the expression of CYP reductase in neuronal cultures further suggests that hepatic CYP-related metabolism may be affected by the expression of mutant human amyloid precursor protein in these tg(hAPP) mice. Therefore, the current study was conducted to investigate the activity and protein content of several CYP isoforms in the livers and kidneys of aged (20-month-old) tg(hAPP) mice. tg(hAPP) mice exhibit significant elevations in hepatic CYP2B, CYP2E1-, CYP3A- and CYP4A-associated activities and CYP4A immunoreactive protein compared with wild-type. In contrast to the liver, a significant depression in renal CYP2E1- and CYP4A-associated activities were demonstrated in tg(hAPP) mice. The presence of the mutant hAPP protein was detected in the brain, kidney and livers of tg(hAPP) mice.
Subject(s)
Alzheimer Disease/enzymology , Cytochrome P-450 Enzyme System/metabolism , Kidney/metabolism , Liver/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Disease Models, Animal , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Hippocampus/metabolism , Humans , Isoenzymes/metabolism , Male , Mice , Mice, Transgenic , Microsomes, Liver/enzymologyABSTRACT
The purpose of this study was to determine if changes in nuclear protein binding of hepatocyte nuclear factor 1 (HNF-1) occur after lipopolysaccharide (LPS) administration. In addition, the time-course of alterations in CYP2E1 regulation were evaluated. Rats were injected with 2.0 mg LPS and euthanized over a 72-h period. Nuclear protein binding to a consensus HNF-1 oligonucleotide was assessed by the electrophoretic mobility shift assay. CYP2E1 activity was analysed using chlorzoxazone as a substrate (60H-CLZ), and CYP2E1 protein concentration was determined by enzyme-linked immunosorbent assay. Endotoxin treatment resulted in decreased nuclear protein binding to an HNF-1 element as early as 1 h after treatment and returned to control levels by 72 h. This reduced binding persisted for 24 h and returned to control values 48 h after LPS administration. In addition, the reduction in binding was primarily attributable to a HNF-1alpha immunoreactive protein. The observed reduction in HNF-1 binding was followed in the time-course by decreases in CYP2E1 activity and protein content with maximal decreases to 50 and 67% of control, respectively, at 48 h after LPS administration. Endotoxin is a potent inducer of the acute phase response (APR). The APR stimulation by endotoxin administration reduced HNF-1alpha binding and decreased the expression of CYP2E1 in the rat liver. The time-course of alterations in HNF-1 and CYP2E1 lend support to the possibility that HNF-1alpha may play a role in the down-regulation of genes that require HNF-1alpha for their constitutive expression. These data serve as an important precedent for future studies evaluating the direct association of decreased HNF-1alpha binding and reduced gene expression after LPS administration.
Subject(s)
Cytochrome P-450 CYP2E1/biosynthesis , DNA-Binding Proteins , Endotoxins/pharmacology , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Cell Nucleus/chemistry , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Enzymologic/drug effects , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Lipopolysaccharides/pharmacology , Liver/drug effects , Liver/enzymology , Male , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Protein Binding , Rats , Rats, Sprague-DawleyABSTRACT
Hepatic cytochrome P450 (CYP) expression and antioxidant activity have been shown to decrease following endotoxin (lipopolysaccharide [LPS]) or proinflammatory cytokine administration. Using mice deficient in interleukin-6 (IL-6), the role of IL-6 in the regulation of hepatic CYP activity, glutathione (GSH) metabolism, and catalase (CAT) activity was analyzed after LPS administration. Administration of LPS produced comparable decreases in hepatic CYP3A activity in WT B6x129 (WT) mice and IL-6 knockout mice. No decrease was observed for CYP2D9 activity after LPS administration in either WT or IL-6 knockout mice. LPS administration significantly increased hepatic and renal CYP2E1 and CYP4A activity in WT mice, with no effect in IL-6 knockout mice. CYP2A12 activity increased in IL-6 knockout, mice with no change in WT mice after LPS administration. LPS administration had no significant effect on hepatic GSH reductase, GST peroxidase, GSH-S-transferase (GST), or total GSH in either WT or IL-6 knockout. However, hepatic CAT activity was significantly reduced in WT mice after LPS administration, with no effect in IL-6 knockout mice. These results support IL-6 as a critical mediator of the effects of LPS on specific hepatic and renal CYP activities and hepatic CAT activity.
Subject(s)
Acute-Phase Reaction/enzymology , Cytochrome P-450 Enzyme System/metabolism , Interleukin-6/genetics , Interleukin-6/physiology , Liver/enzymology , Animals , Antioxidants/metabolism , Catalase/metabolism , Glutathione/metabolism , Kidney/enzymology , Lipopolysaccharides/pharmacology , Liver/drug effects , Mice , Mice, Knockout , Microsomes, Liver/enzymologyABSTRACT
The promoting effects of polychlorinated biphenyls (PCBs) have been studied extensively in a variety of two-stage carcinogenesis models. However, the molecular mechanisms responsible for the promotion effects of PCBs have not been elucidated. We measured the effect of PCBs on DNA-binding proteins involved in cell proliferation and transformation. Male Sprague-Dawley rats were injected intraperitoneally with mono-, di-, tri-, tetra-, or hexachlorobiphenyls (300 micromol/kg/d) each day for 4 d and killed 4 h after the last injection. To detect alterations in nuclear proteins that could explain the tumor-promoter activity of PCBs, liver nuclear extracts were analyzed by electrophoretic mobility shift assays. Electrophoretic mobility shift assay analysis of signal transducers and activators of transcription (STAT)-binding activity to a consensus gamma-interferon-activated sequence (GAS) element was compared in liver nuclear extracts from treated rats. STAT-binding activity was eightfold to tenfold higher in nuclear extracts from animals treated with 2,4,4'-trichloro- (PCB 28) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Analysis of the protein complex binding to the GAS element, with antibodies specific for STAT3, STAT5, and STAT6, indicated that the protein complex was made up of STAT5 and STAT6 proteins. HepG2 cells transiently transfected with a luciferase reporter gene construct containing many STAT5 binding sites were treated with PCB 28 and PCB 153. PCB 28 stimulated a greater than 25-fold increase in luciferase activity at the highest concentration tested, 1.0 microg/mL. However, enhanced luciferase activity did not occur with PCB 153 treatment. 4-Chlorobiphenyl (PCB 3), PCB 28, and PCB 153 treatment of Sprague-Dawley rats resulted in a large increase in protein binding to a consensus activated protein-1 (AP-1) element. However, 3,4-dichlorobiphenyl (PCB 12) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) treatments did not increase AP-1 transcription activity. Further analysis of the proteins binding to the AP-1 consensus sequence with antibodies specific for c-fos, junD, and junB indicated that the protein composition consists of junD proteins. These data showed functional differences between noncoplanar and coplanar PCBs with respect to STAT activation and AP-1-DNA binding.
Subject(s)
DNA-Binding Proteins/genetics , Milk Proteins , Polychlorinated Biphenyls/pharmacology , Trans-Activators/genetics , Animals , Cytochrome P-450 Enzyme System/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , Electrophoresis, Agar Gel , Liver/metabolism , Luciferases/metabolism , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , STAT5 Transcription Factor , Trans-Activators/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolismABSTRACT
Interest in the non-neuronal alterations following traumatic brain injury (TBI) has led to research evaluating hepatic metabolism following injury. Several models of injury demonstrate tissue-specific alterations in cytochrome P450 activity. This study examined tissue-specific alterations in cytochrome P450-mediated hydroxylation in the rat model of TBI. Male Sprague-Dawley rats received anesthesia alone, craniotomy, or craniotomy plus TBI. Rats were sacrificed at 24 and 48 h. Liver, kidney, and brain cortex microsomes were isolated. Total liver P450 content, 6-hydroxychlorzoxazone formation rate, and CYP2E1 protein were evaluated. In liver microsomes, spectral P450 was decreased to 86 +/- 5% (p < 0.05) of control at 24 h following injury, and 6-hydroxychlorzoxazone formation rate decreased to 74 +/- 18% of control (p < 0.05) at 48 h following injury. In kidney microsomes, 6-hydroxychlorzoxazone formation rate was increased to 154% of control (p < 0.01) 24 h following injury. 6-Hydroxychlorzoxazone formation rate was unaffected by TBI in brain cortical microsomes. The CYP2E1 inhibitor, 4-methylpyrazole, inhibited the formation of 6-hydroxychlorzoxazone in brain, kidney, and liver microsomes. These data demonstrate that tissue-specific alterations in 6-hydroxychlorzoxazone formation rate occur following TBI.
Subject(s)
Brain Injuries/metabolism , Brain/pathology , Chlorzoxazone/analogs & derivatives , Chlorzoxazone/metabolism , Animals , Brain/enzymology , Brain/metabolism , Brain Injuries/enzymology , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 CYP2E1 Inhibitors , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Fomepizole , Hydroxylation , Kidney/enzymology , Kidney/metabolism , Liver/enzymology , Liver/metabolism , Male , Microsomes/enzymology , Microsomes/metabolism , Organ Specificity , Pyrazoles/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: Inflammation induced by Escherichia coli lipopolysaccharide alters the clearance of several hepatically eliminated drugs. Extensive rat liver research has shown CYP2E1 down-regulation after lipopolysaccharide administration. To further investigate this phenomenon in humans, lipopolysaccharide was administered to healthy male volunteers and chlorzoxazone was used as a CYP2E1 probe drug. METHODS: Twelve healthy men were given 500 mg oral chlorzoxazone after two daily lipopolysaccharide doses (20 endotoxin units/kg/day) and again after administration of saline solution in this balanced crossover study. Serum and urine chlorzoxazone and 6-hydroxychlorzoxazone were quantified, as well as cytokine and C-reactive protein levels. RESULTS: Lipopolysaccharide produced the expected induction of the acute-phase response shown by elevations in tumor necrosis factor, interleukin-6, C-reactive protein, and temperature. Lipopolysaccharide treatment failed to produce a significant change in the chlorzoxazone oral clearance (4.4 +/- 0.9 mL/min/kg for lipopolysaccharide versus 4.2 +/- 1.4 mL/min/kg for control) or the 6-hydroxychlorzoxazone formation clearance (2.8 +/- 0.65 mL/min/kg for lipopolysaccharide versus 2.5 +/- 0.9 mL/min/kg for control). The high intersubject variabilities in oral clearance and formation clearance were not accounted for by changes in protein binding, cytokine, or C-reactive protein values. In contrast, a significant increase in the 6-hydroxychlorzoxazone glucuronide renal clearance was observed (7.5 +/- 1.37 mL/min/kg for lipopolysaccharide versus 6.1 +/- 1.7 mL/min/kg for control). CONCLUSIONS: This study showed that the inflammatory response to lipopolysaccharide (20 endotoxin units/kg/day for 2 days) in humans does not consistently alter chlorzoxazone hepatic metabolism. However, the significant increase in renal clearance of the glucuronidated metabolite suggests that renal tubular secretion may be increased in humans with acute endotoxemia.
Subject(s)
Chlorzoxazone/pharmacokinetics , Cytochrome P-450 CYP2E1/metabolism , Lipopolysaccharides/adverse effects , Muscle Relaxants, Central/pharmacokinetics , Adult , C-Reactive Protein/metabolism , Chlorzoxazone/analogs & derivatives , Chlorzoxazone/blood , Chlorzoxazone/urine , Humans , Interleukin-6/blood , Kidney Tubules/metabolism , Lipopolysaccharides/administration & dosage , Liver/metabolism , Male , Muscle Relaxants, Central/blood , Muscle Relaxants, Central/urine , Reference Values , Serum Albumin/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
STUDY OBJECTIVE: To assess the effect of recombinant human growth hormone (rhGH) on the insulin-like growth factor-1 (IGF-1) plasma concentration versus time profile during continuous infusion of recombinant human (rh)IGF-1 to patients with traumatic brain injury (TBI). SETTING: University of Kentucky Chandler Medical Center. PATIENTS: Twenty-three patients with TBI (aged 18-59 yrs) with Glascow Coma Scale scores of 4-10. INTERVENTION: Patients were randomized to receive rhIGF-1 0.01 mg/kg/hour and daily subcutaneous doses of rhGH 0.05 mg/kg/day or saline for 14 days. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of IGF-1 and IGF-binding protein (BP)-3 were quantified by radioimmunoassay. Patients receiving rhIGF-1/rhGH reached a peak IGF-1 concentration (1199.3+/-84.0 microg/L) at 72 hours and maintained it throughout the study. Levels of IGF-1 in the control group did not change significantly above baseline throughout the study. Concentrations of IGFBP-3 were significantly higher after 48 hours in the treated group (5.1+/-0.4 mg/L) than in controls (2.9+/-0.5 mg/L) and continued until the end of the study (p<0.05). CONCLUSION: Infusion of rhIGF-1 in conjunction with rhGH effectively achieved and maintained supraphysiologic IGF-1 plasma concentrations throughout the dosing period in patients with TBI. It appears that rhGH alters the IGF-1 plasma concentration versus time profile during continuous administration. Although speculative, changes in protein binding of IGF-1 are the most likely mechanism.
Subject(s)
Brain Injuries/blood , Human Growth Hormone/pharmacology , Insulin-Like Growth Factor Binding Protein 3/blood , Insulin-Like Growth Factor I/pharmacology , Adolescent , Adult , Human Growth Hormone/administration & dosage , Humans , Insulin-Like Growth Factor I/metabolism , Middle Aged , Radioimmunoassay , Recombinant Proteins , Time FactorsABSTRACT
The purpose of this study was to evaluate the effect of pregnenolone 16 alpha-carbonitrile (PCN) on the interconversion pharmacokinetics and metabolism of dapsone. To determine microsomal CYP3A activity and protein, eight rats (4 PCN, 4 corn oil) received a 1 mg kg(-1) intravenous bolus dose of dapsone, followed by blood and urine sampling. The formation clearance of dapsone hydroxylamine (CLf DDS-NOH) was calculated from the obtained samples. Interconversion pharmacokinetics estimates were obtained after 10 rats (5 PCN, 5 control) received 1 mg kg(-1) dapsone or 1.17 mg kg(-1) monoacetyldapsone, with a 24-h wash-out. Results from the interconversion analysis demonstrated that PCN significantly increased systemic clearance (CLs) of dapsone, but not its interconversion. The in-vivo/in-vitro correlation study demonstrated that PCN significantly increased CLs of dapsone (8.55 to 16.39mLmin(-1); P<0.01) and CLf DDS-NOH (0.13 to 0.18mLmin(-1); P<0.01). PCN treatment produced a 69% increase in CYP3A protein, and increased 6beta- and 2beta-hydroxytestosterone formation rates. Significant correlations were found between CLf DDS-NOH and either 6beta- (r2 = 0.925), 2beta-hydroxytestosterone (r2 = 0.92), or CYP3A1/2 protein (r2= 0.60). We conclude that PCN treatment produces significant increases in CLs (dapsone) and CLf (DDS-NOH) in rats. These changes were not due to changes in the reversible metabolism of dapsone. These results suggest that the formation clearance of dapsone hydroxylamine reflects alterations in CYP3A activity, despite the fact that it accounted for a small part of the systemic clearance of dapsone.
Subject(s)
Anti-Infective Agents/pharmacokinetics , Aryl Hydrocarbon Hydroxylases , Dapsone/pharmacokinetics , Pregnenolone Carbonitrile/pharmacology , Animals , Anti-Infective Agents/metabolism , Area Under Curve , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/metabolism , Dapsone/analogs & derivatives , Dapsone/blood , Dapsone/metabolism , Half-Life , Injections, Intravenous , Male , Metabolic Clearance Rate , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Oxidoreductases, N-Demethylating/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Leptin is a hormone that is secreted by adipocytes and regulates body weight through its effect on satiety and energy metabolism. The ob/ob mouse is deficient in this protein and is characterized by obesity and other metabolic disorders. This study investigated the alterations of several hepatic cytochrome P-450 (CYP), conjugation, and antioxidant enzymes in lean and ob/ob mice and the role leptin plays in the modulation of these enzymes. Lean and ob/ob male mice were injected with leptin (100 microg) or PBS for 15 days. Liver microsomes from ob/ob mice, when compared with lean controls, displayed significantly reduced chlorzoxazone 6-hydroxylation activity (27%); however, 7alpha- and 16alpha- testosterone hydroxylation and pentoxyresorufin O-dealkylation activities were significantly higher (47%, 22%, and 39%, respectively). Leptin administration corrected alterations seen with all P-450 activities. Dealkylation of ethoxyresorufin and omega-hydroxylation of lauric acid activities from ob/ob and lean mice were not statistically different; however, leptin exposure significantly increased ethoxyresorufin activity in lean mice (14%) and decreased the activity in ob/ob mice (36%). UDP-glucuronosyl-transferase and glutathione S-transferase activities were not altered. The antioxidant enzymes, catalase (11%) and glutathione peroxidase (26%), as well as glutathione reductase (17%), were lower in the ob/ob mice and leptin treatment corrected these alterations. The results of this study demonstrate alterations in constitutive expression of CYP2B, CYP2E, CYP2A, catalase, glutathione peroxidase, and glutathione reductase in ob/ob mice that were restored to lean control values following leptin treatment. Additionally, CYP3A activity was increased following leptin treatment in ob/ob mice. The mechanism for the observed alterations may be due to direct leptin effects or via indirect alterations in insulin, corticosterone, and/or growth hormone.
Subject(s)
Antioxidants/metabolism , Cytochrome P-450 Enzyme System/metabolism , Liver/enzymology , Proteins/pharmacology , Animals , Blood Glucose/metabolism , Body Weight , Corticosterone/blood , Cytosol/enzymology , Cytosol/metabolism , Glucuronosyltransferase/metabolism , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Hydroxylation , Insulin/blood , Leptin , Male , Mice , Mice, Obese , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Testosterone/metabolismABSTRACT
Hepatic cytochromes P-450 (CYP) are well characterized drug and xenobiotic metabolizing enzymes that are extensively regulated by genetic and environmental factors. Inflammatory mediators, including interleukins (ILs), interferons (IFNs), and tumor necrosis factor-alpha (TNF-alpha), have been shown to down-regulate several CYP isoforms; however, elucidation of the inflammatory mediators that are responsible for specific CYP down-regulation is difficult. The purpose of this experiment was to evaluate the role endogenous TNF-alpha plays in the regulation of liver CYP expression after endotoxin administration. Mice deficient in the p55 and p75 TNF receptors and wild-type mice were given Gram-negative bacterial lipopolysaccharide (LPS) and killed 24 h after administration. CYP analysis indicates that LPS decreases CYP1A, CYP2B, CYP3A, and CYP4A independently of TNF-alpha. CYP2D9 and CYP2E1 activities show differential responses to LPS between wild-type and TNF p55/p75 receptor knockout mice, indicating the down-regulation of CYP2D9 and CYP2E1 is differentially modulated by TNF-alpha expression. Furthermore, TNF-alpha appears to affect the constitutive expression of CYP2D9 and CYP2E1. To date, this is the first evidence suggesting that a proinflammatory cytokine is involved in the constitutive regulation of drug-metabolizing enzymes.
Subject(s)
Cytochrome P-450 Enzyme System/biosynthesis , Isoenzymes/biosynthesis , Lipopolysaccharides , Liver/enzymology , Receptors, Tumor Necrosis Factor/deficiency , Animals , Down-Regulation , Liver/drug effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Receptors, Tumor Necrosis Factor/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S-transferase (GST), and UDP-glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague-Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S-transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin-treated animals. Changes in the conjugative enzymes and the free-radical scavenging enzymes following endotoxin exposure may alter the host's metabolism and response to free radicals.
Subject(s)
Endotoxins/toxicity , Microsomes, Liver/drug effects , Animals , Catalase/metabolism , Escherichia coli/metabolism , Glucuronosyltransferase/metabolism , Glutathione Peroxidase/metabolism , Glutathione Transferase/metabolism , Lipid Peroxidation , Male , Microsomes, Liver/enzymology , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolismABSTRACT
There is evidence to suggest that obese populations have an increased susceptibility to various pathologic disorders. Both AP-1 and STAT nuclear binding proteins have been suggested to play a role in certain obesity-related diseases. The objective of our studies reported herein was to compare constitutive binding activity of nuclear proteins (AP-1, GR, and STAT), that may be relevant to obesity-related diseases in the obese (fa/fa) Zucker rat to lean (Fa/?) littermates. AP-1, GR, and STAT liver nuclear protein binding activity was analyzed using the electrophoretic mobility shift assay (EMSA). EMSA analysis of liver nuclear protein from obese and lean Zucker rats revealed high constitutive AP-1 binding activity in the obese animals. AP-1 binding activity in the obese rats was not further elevated by treatment with phenobarbital, a known inducer of AP-1 binding activity. No differences were observed in GR binding to a consensus GRE between obese and lean animals; however, STAT binding activity to a consensus GAS element was lower in liver tissue from obese Zucker rats. Our findings presented herein suggest that the fa/fa Zucker rat may be a suitable obese rodent model for studying the roles AP-1 and STAT may play in the pathologies of these diseases.
Subject(s)
DNA-Binding Proteins/metabolism , Liver/metabolism , Obesity/genetics , Receptors, Glucocorticoid/metabolism , Trans-Activators/metabolism , Transcription Factor AP-1/metabolism , Animals , Cell Nucleus/metabolism , Disease Models, Animal , Electrophoresis, Polyacrylamide Gel , Liver/cytology , Liver Extracts/metabolism , Male , Obesity/metabolism , Rats , Rats, Zucker , Receptors, Glucocorticoid/genetics , Signal TransductionABSTRACT
The levels of degradation of cefetamet pivoxil (CAT), cefuroxime axetil (CAE), and cefpodoxime proxetil (CPD) in 0.6 M phosphate buffer (pH 7.4) and human intestinal juice (pH 7.4) at 37 degreesC over 24 h were compared. Significant differences in the time courses of degradation and in the patterns of degradation products were observed. (i) The relative proportions of the Delta2- and Delta3-cephalosporins were roughly reversed in the two incubation media. In phosphate buffer, the major degradation product was the Delta2-cephalosporin (CAT = 61%; CAE = 74%; CPD = 85%), while in intestinal juice it was the Delta3-cephalosporin (CAT = 86%; CAE = 75%; CPD = 87%). (ii) Generally, the degradation of the prodrug esters progressed faster in intestinal juice than in phosphate buffer (e.g., for CAT the half-lives [t1/2s] were 0.78 and 4.3 h, respectively). (iii) The two diastereoisomers of CAE and CPD were degraded at different rates in intestinal juice (for the CAE diasteroisomers, t1/2s = 0.37 and 0.93 h; for the CPD diastereoisomers, t1/2s = 0.18 and 0.98 h) but were degraded at similar rates in phosphate buffer (for the CAE diastereoisomers, t1/2 = 1.6 h; for the CPD t1/2 diastereoisomers, = 2.2 h). It is concluded that (i) the Delta2 isomerization does not significantly affect the bioavailability of prodrug esters since enzymatic hydrolysis in the intestinal fluid proceeds mainly to the active Delta3-cephalosporin and (ii) the high degree of stereoselectivity of the enzymatic ester hydrolysis should make it possible to increase the bioavailabilities of certain prodrug esters (CAE, CPD) by using the more stable diasterioisomer.
Subject(s)
Cephalosporins/pharmacokinetics , Intestinal Mucosa/metabolism , Prodrugs/pharmacokinetics , Administration, Oral , Adult , Biological Availability , Ceftizoxime/analogs & derivatives , Ceftizoxime/pharmacokinetics , Cefuroxime/analogs & derivatives , Cefuroxime/pharmacokinetics , Drug Stability , Humans , Male , Cefpodoxime ProxetilABSTRACT
PURPOSE: The purpose of our research was two-fold: 1) to further characterize the downregulation of CYP3A2 mRNA, protein, and activity during an acute phase response (APR); 2) most importantly, to relate the time-dependent activation of nuclear proteins to putative DNA binding sequences within the CYP3A2 5'-flanking region, with the loss in CYP3A2 expression. METHODS: Rats were injected (2.0 mg/animal, i.p.) with LPS and sacrificed at 1, 2, 4, 6, 8, 24, 48, and 72 hours. Hepatic nuclear protein was isolated and analyzed for binding activity to AP-1, NFkappaB, and NF-IL6 consensus sequences. Hepatic CYP3A2 mRNA levels were determined by solution hybridization and CYP3A2 protein, CYP3A2 activity, and total P450 were measured in hepatic microsomes. RESULTS: Computer analysis of the 5'-flanking region of CYP3A2 revealed the presence of 5 NF-IL6 and 4 AP-1 putative DNA binding sites. The strongest increase in AP-1 binding activity occurred between 6 and 24 hr, and the alteration in binding complexes to an NF-IL6 oligonucleotide occurred between 4 and 24 hr. Maximum loss in CYP3A2 mRNA occurred at 8 hr post-LPS injection and remained lowered at the 24 hr timepoint. CYP3A2 protein was significantly decreased at 24, 48, and 72 hours post-LPS treatment with corresponding decreases in CYP3A2 activity and total P450. CONCLUSIONS: The changes in NF-IL6 and AP-1 binding after LPS treatment, which appears to correlate with the changes in CYP3A2 mRNA, combined with the presence of putative NF-IL6 and AP-1 sites located in the CYP3A25'-flanking region, may indicate a potential role for NF-IL6 and AP-1 in CYP3A2 downregulation during an APR.
Subject(s)
Cytochrome P-450 Enzyme System/drug effects , Lipopolysaccharides/pharmacology , Steroid Hydroxylases/drug effects , Acute-Phase Reaction/metabolism , Animals , CCAAT-Enhancer-Binding Proteins , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , DNA-Binding Proteins/metabolism , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Male , NF-kappa B/metabolism , Nuclear Proteins/metabolism , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Steroid Hydroxylases/genetics , Steroid Hydroxylases/metabolism , Transcription Factor AP-1/metabolismABSTRACT
AIMS: In men, the inflammatory response to intravenous endotoxin depresses apparent oral clearances of antipyrine, hexobarbitone, and theophylline. The aim of this study was to investigate whether there might be gender differences in the regulation of hepatic cytochromes P450. METHODS: Experiments were carried out in seven healthy women volunteers (ages 19-51, median 22 years). Each woman received a cocktail of the three drugs on two occasions, once after a saline injection and again after endotoxin. RESULTS: Endotoxin injections, but not saline, caused the expected physiologic responses of inflammation including fever and increases in circulating tumor necrosis factor-alpha, interleukin-6, and C-reactive protein. When compared with the saline control studies, endotoxin significantly decreased clearances of all probes: antipyrine, 31% (95%CI 21%-41%); hexobarbitone, 20% (95%CI 10-31%); and theophylline, 20% (95%CI 10%-30%). The decreases were comparable with those found in the men previously studied (35%, 27%, and 22%, respectively). CONCLUSIONS: These data show that endotoxin-induced inflammation decreases hepatic cytochrome P450-mediated metabolism of selected probe drugs in women as it does in men.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Antipyrine/pharmacokinetics , Cytochrome P-450 Enzyme System/metabolism , Hexobarbital/pharmacokinetics , Hypnotics and Sedatives/pharmacokinetics , Lipopolysaccharides/adverse effects , Liver/enzymology , Phosphodiesterase Inhibitors/pharmacokinetics , Theophylline/pharmacokinetics , Adult , Anti-Inflammatory Agents, Non-Steroidal/blood , Antipyrine/blood , Area Under Curve , C-Reactive Protein/analysis , Female , Fever/chemically induced , Hexobarbital/blood , Humans , Hypnotics and Sedatives/blood , Interleukin-6/blood , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/metabolism , Liver/drug effects , Middle Aged , Nephelometry and Turbidimetry , Phosphodiesterase Inhibitors/blood , Regression Analysis , Theophylline/blood , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Amantadine is an antiviral agent effective against influenza A viruses. We investigated 1) the antiviral efficacy, 2) analytical detection, 3) bioavailability and disposition, 4) pharmacokinetic modelling and 5) adverse reactions of amantadine in the horse. In vitro, amantadine and its derivative rimantadine suppressed the replication of recent isolates of equine-2 influenza virus with effective doses (EDs) of less than 30 ng/ml. Rimantadine was more effective than amantadine against most viral isolates; we suggest a minimum plasma concentration of 300 ng/ml of amantadine for therapeutic efficacy. In vivo an i.v. dose of amantadine 15 mg/kg bwt produced mild, transient CNS signs which were no longer apparent after 30 min. Amantadine administered at a dose of 15 mg/kg bwt was established as the maximum safe single i.v. dose. However, if repeated i.v. administration of amantadine is required no more than 10 mg/kg bwt t.i.d. should be used. The maximal safe plasma concentration of amantadine was not evaluated but is probably greater than 2000 ng/ml and possibly greater than 4000 ng/ml. On the other hand, horses with lower seizure thresholds, or those on medications that lower seizure thresholds, may be at increased risk of amantadine-induced seizures, which show few premonitory signs and are rapidly fatal. After i.v. administration of amantadine 10 mg/kg bwt, the disposition kinetics were well fitted by a 2-compartment open model. The estimated peak plasma concentration after this dose was about 4500 ng/ml, the volume of distribution at steady-state (Vdss) was (mean +/- s.d.) 4.9 +/- 1.9 l/kg bwt and the beta phase half-life was 1.83 +/- 0.87 h. Computer projections of plasma amantadine concentrations after i.v. administration of amantadine at a dose of 10 mg/kg bwt t.i.d. at 8 h intervals suggest peak plasma concentrations of 4000-5000 ng/ml and troughs of less than 300 ng/ml will be achieved. Amantadine administered orally at 10 mg/kg bwt and 20 mg/kg bwt showed mean oral bioavailability of about 40-60% and a plasma half life of 3.4 +/- 1.4 h; however, there was substantial inter-animal variation in bioavailability. Projections based on the kinetics observed in individual animals suggest that some animals readily maintain effective plasma concentrations of amantadine after oral administration of 20 mg/kg bwt t.i.d. On the other hand, animals in which amantadine is poorly bioavailable may require up to a 6-fold (120 mg/kg bwt) increase in the oral dose to achieve effective blood concentrations. Withholding food for 15 h did not reduce these inter-animal differences in bioavailability. Our results showed that simple dosing with oral amantadine will not yield effective plasma concentrations in all animals. While i.v. administration yielded more reproducible plasma concentrations, care should be taken to see that the seizure threshold is not exceeded. In acute situations, i.v. administration (5 mg/kg bwt) every 4 h should maintain safe and effective plasma and respiratory tract concentrations of amantadine.
Subject(s)
Amantadine/pharmacology , Amantadine/pharmacokinetics , Antiviral Agents/pharmacology , Antiviral Agents/pharmacokinetics , Central Nervous System/drug effects , Horses/physiology , Administration, Oral , Amantadine/blood , Animals , Antiviral Agents/adverse effects , Biological Availability , Central Nervous System/physiology , Chromatography, Gas/methods , Chromatography, Gas/veterinary , Dose-Response Relationship, Drug , Female , Horse Diseases/drug therapy , Horse Diseases/physiopathology , Horse Diseases/prevention & control , Influenza A virus/drug effects , Injections, Intravenous , Mass Spectrometry/methods , Mass Spectrometry/veterinary , Orthomyxoviridae Infections/drug therapy , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/veterinary , Seizures/chemically induced , Seizures/veterinary , Time FactorsABSTRACT
OBJECTIVES: This study examined factors that affect cost, reliability, and the value of determining the cytochrome P450 2D6 (CYP2D6) polymorphism in clinical practice. STUDY DESIGN: The method of deoxyribonucleic acid isolation, sample preparation, oligonucleotide primers, and polymerase chain reaction procedures were scrutinized for their effect on CYP2D6 genotyping efforts. The determination of the CYP2D6 A, B, D, E, and T alleles was used to identify the deficiency in CYP2D6 expression in 161 individuals phenotyped for CYP2D6 activity with dextromethorphan. The CYP2D6 genotype was assessed in 74 outpatients who had received diagnoses of depression. Eighteen of these patients were screened because of an adverse response to a tricyclic or antidepressant known or suspected to be a CYP2D6 substrate. RESULTS: The CYP2D6 A, B, C, D, E, and T alleles could be detected in 13 hours at a cost of $84 per sample by judicious selection of conditions and procedures. The genotype provided an accurate predictor of CYP2D6 expression in all 134 subjects who expressed the enzyme and in all 27 unrelated individuals phenotyped as deficient in CYP2D6 activity. In the patient group that experienced adverse effects, 44% of all CYP2D6 gene copies contained the A, B, D, E, or T allele(s) associated with inactive CYP2D6 expression. This was more than twice the rate for the occurrence of mutant alleles in the other 56 psychiatric patients (21%) and in 80 random subjects from the general population (20%; p < 0.05). CONCLUSIONS: Screening psychiatric patients for CYP2D6 expression may distinguish metabolic-based therapeutic problems from drug sensitivity caused by other mechanisms.
Subject(s)
Cytochrome P-450 CYP2D6/genetics , DNA/isolation & purification , Depression/enzymology , Genotype , Polymorphism, Genetic/genetics , Antidepressive Agents, Tricyclic/therapeutic use , Depression/drug therapy , Genetic Testing/economics , Humans , Polymerase Chain Reaction/standards , Quality Control , Reagent Kits, Diagnostic/standards , Reproducibility of ResultsABSTRACT
Phenobarbital (PB) is a potent inducer of cytochrome P450 enzymes, particularly CYP2B1/2B2. Although the mechanism(s) of PB induction of CYP2B1/2B2 is not fully understood, current research is focusing on the role PB may play in altering the binding of nuclear proteins to critical DNA response elements in the 5'-flanking region of these genes. In this study, rat liver nuclear proteins were analyzed for DNA binding ability using both a general consensus and a CYP2B2 sequence-specific AP-1 oligonucleotide. We demonstrate that in vivo PB treatment enhances protein binding activity to the consensus AP-1 oligonucleotide. Likewise, a putative AP-1 site, identified at -1441 in the CYP2B2 5'-flanking region, also formed a sequence specific DNA/protein complex which was enhanced after PB exposure. These data may support a role of AP-1 in the PB induction mechanism of CYP2B1/2B2.