Biomedical subjects

D F Lewis

Publications and source records attributed to D F Lewis.

At least 19 recordsLinked to original sources

PubMed2001

Synthesis of dixanthones and poly(dixanthone)s by cyclization of 2-aryloxybenzonitriles in trifluoromethanesulfonic acid.

[reaction: see text] Condensation of 2-fluorobenzonitriles with phenoxides affords 2-aryloxybenzonitriles that cyclize cleanly in trifluoromethanesulfonic acid at room temperature to give xanthone-iminium triflates. The C=N bond in these compounds is remarkably resistant to hydrolysis, but prolonged reaction with strong aqueous acid under vigorous conditions affords xanthones in good yield. The synthesis is exemplified for a novel series of polynuclear dixanthones and for a high molar mass polyxanthone derived from the previously unreported monomer 3,3'-difluoro-4,4'-biphenyldicarbonitrile.

Journal Article↗

PubMed2001

Application of process chemistry and SAR modelling to the evaluation of health findings of lower olefins.

Epidemiology studies show increased leukemia mortality among styrene butadiene rubber (SBR) workers but not among butadiene monomer production employees. A detailed review of the SBR manufacturing process indicates that sodium dimethyldithiocarbamate (DMDTC) introduced into the SBR manufacturing process for a period in the 1950s coincides with increased leukemia mortality. Using the Computer-Optimized Molecular Parametric Analysis of Chemical Toxicity (COMPACT), we assessed the enzyme (cytochrome P450) substrate specificity of an olefin series including 1,3-butadiene (BD) and also modeled its interaction with DMDTC. These analyses showed correlation of a structural/electronic parameter--the COMPACT radius--with the presence or absence of cytogenetic activity and also found that DMDTC would inhibit the oxidative metabolism of BD at least at high concentrations. Both DMDTC and its diethyl analog (DEDTC) bind with CYP 2E1 and CYP 2A6. Both of these isoforms are important in the initial oxidative metabolism of butadiene and other olefins. In co-exposure studies in mice of DMDTC with BD or with epoxybutene (EB), we found that there was a reduced increase in genotoxic activity based on micronuclei induction compared with BD or EB exposure alone. Treatment with DMDTC significantly increased the protein carbonyl contents of hepatic microsomes compared with that of controls, a finding that may be related to DMDTC's activity as a prooxidant. Co-exposure with DMDTC and EB increased hepatic microsomal carbonyls to levels significantly greater than those of DMDTC-treated mice, while EB administration in the absence of DMDTC did not change protein carbonyls relative to those of controls. The increase in hepatic microsomal protein carbonyls suggests that DMDTC may modulate EB metabolism towards the formation of reactive intermediates that react with proteins. The present molecular modeling and mechanistic studies suggest that co-exposure of BD and DMDTC is a plausible biological hypothesis regarding increased leukemia risk among SBR workers.

Alkenes↗

PubMed2001

Tocolytic therapy with preterm premature rupture of membranes.

Tocolytic therapy with preterm premature rupture of membranes continues to be a controversial issue. Articles focusing on potential benefits and adverse outcomes associated with tocolysis in this disease process are reviewed. Although there may be some short-term neonatal benefit, further investigation of this issue is warranted.

Antibiotic Prophylaxis↗

PubMed2001

Contribution of CYP1A2 in the hepatic metabolism of melatonin: studies with isolated microsomal preparations and liver slices.

The objective of the present studies was to define the enzyme systems catalysing the 6-hydroxylation of melatonin, by monitoring the levels of 6-sulphatoxymelatonin in rat hepatic postmitochondrial preparations and in precision-cut liver slices. Melatonin 6-hydroxylase activity was localized in microsomes and was supported by NADPH, but not NADH. Treatment of rats with beta-naphthoflavone more than tripled 6-sulphatoxymelatonin formation from melatonin, but gave rise only to a moderate increase (25%) in the sulphate conjugation of 6-hydroxymelatonin. Treatment of rats with phenobarbitone, acetone, dexamethasone and clofibrate did not increase 6-sulphatoxymelatonin generation when either melatonin or 6-hydroxymelatonin served as substrates. Of a number of cytochrome P450 inhibitors investigated, only furafylline inhibited markedly the conversion of melatonin to 6-sulphatoxymelatonin without any concomitant effect on the sulphoconjugation of 6-hydroxymelatonin. When liver slices were incubated with melatonin, treatment of rats with beta-naphthoflavone, and to a lesser extent phenobarbitone, elevated the levels of 6-sulphatoxymelatonin in the culture medium. No such increase was seen when slices from beta-naphthoflavone-treated rats were incubated with 6-hydroxymelatonin, whereas a modest increase was seen with slices from phenobarbitone-treated rats. Treatment of rats with acetone, dexamethasone or clofibrate failed to modulate the levels of 6-sulphatoxymelatonin generated from either melatonin or 6-hydroxymelatonin. Molecular modelling analysis revealed that melatonin had a high area/depth(2) ratio, displayed characteristics of CYP1A2 substrates and could be readily accommodated into the human CYP1A2 active site in a position favouring 6-hydroxylation. Collectively, all the above data provide strong experimental evidence that CYP1A2 is an important catalyst of the 6-hydroxylation of melatonin.

Animals↗

PubMed2001

Allosteric phenomena in cytochrome P450-catalyzed monooxygenations.

Allosteric regulation of monooxygenase activity is shown to occur with diverse cytochrome P450 isoforms and is characterized by kinetic patterns deviating from the Michaelis-Menten model. Homotropic and heterotropic phenomena are encountered in both substrate activation and productive coupling of the electron donors NADPH-cytochrome P450 reductase and cytochrome b5, and the lipid environment of the system also appears to play a role as an effector. Circumstantial analysis reveals the components of the electron transfer chain to be mutually beneficial in interactions with each other depending on the substrate used and type of cytochrome P450 operative. It is noteworthy that association of diatomic gaseous ligands may be amenable to allosteric regulation as well. Thus, dioxygen binding to cytochrome P450 displays nonhyperbolic kinetic profiles in the presence of certain substrates; the latter, together with redox proteins such as cytochrome b5, can exert efficient control of the abortive breakdown of the oxyferrous intermediates formed. Similarly, substrates may modulate the structural features of the access channel for solutes such as carbon monoxide in specific cytochrome P450 isozymes to either facilitate or impair ligand diffusion to the heme iron. The in vivo importance of allosteric regulation of enzyme activity is discussed in detail.

Allosteric Regulation↗

PubMed2001

Detection of interleukin-6 in maternal plasma predicts neonatal and infectious complications in preterm premature rupture of membranes.

The objective of this study is to determine if the detection of interleukin-6 (IL-6) in maternal plasma prior to delivery predicts neonatal and/or infectious complications in patients with preterm premature rupture of membranes. Patients with preterm premature rupture of membranes between 24 and 35 weeks' gestation were asked to participate in the study. Maternal blood was obtained prior to delivery. All patients received Ampicillin-sulbactam and steroids. IL-6 concentrations were determined by enzyme-linked immunoadsorbent assay (ELISA) using 50 mL of plasma assayed in duplicate. ELISA sensitivity was 18 pg/mL. Neonatal and infectious complications examined were respiratory distress syndrome, necrotizing enterocolitis, intraventricular hemorrhage, intra-amniotic infection, presumed neonatal sepsis, neonatal sepsis, and congenital pneumonia. Fifty-seven patients' plasma was analyzed. Thirty-five had positive plasma IL-6 prior to delivery. Twenty-seven patients had at least one neonatal complication with 24 (89%) being positive for IL-6. Of the 30 patients without complications, only 11 (37%) were positive (p = 0.0001, OR 13.8. 95% CI, 2.93-74.7). A subanalysis of patients who received a course of corticosteroids was performed and significance was maintained. Ten of 13 patients (77%) with neonatal complications had positive IL-6 compared with 40% without complications (p <or=0.01). Infectious morbidity occurred in 32 patients with 24 having positive IL-6 values (75%). Only 11 of 25 (44%) without infections were positive (p <or=0.03, OR 3.82, 95%, CI 1.09-13.0). The presence of IL-6 in the maternal plasma predicted patients with neonatal complications. These correlations persisted when the data were stratified for those patients who received corticosteroids. It also predicted infectious complications.

Adult↗

PubMed2001

Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4.

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.

Coumarins↗

PubMed2001

Carbamazepine: a 'blind' assessment of CVP-associated metabolism and interactions in human liver-derived in vitro systems.

1. The ability of various in vitro systems for CYP enzymes (computer modelling, human liver microsomes, precision-cut liver slices, hepatocytes in culture, recombinant enzymes) to predict various aspects of in vivo metabolism and kinetics of carbamazepine (CBZ) was investigated. 2. The study was part of the EUROCYP project that aimed to evaluate relevant human in vitro systems to study drug metabolism. 3. CBZ was given to the participating laboratories without disclosing its chemical nature. 4. The most important enzyme (CYP3A4) and metabolic route (10,11-epoxidation) were predicted by all the systems studied. 5. Minor enzymes and routes were predicted to a different extent by various systems. 6. Prediction of a clearance class, i.e. slow clearance, was correctly predicted by microsomes, slices, hepatocytes and recombinant enzymes (CYP3A4). 7. The 10,11-epoxidation of CBZ by the recombinant CYP3A4 was enhanced by the addition of exogenous cytochrome-b5, leading to a considerable over-prediction. 8. Induction potency of CBZ was predicted in cultured hepatocytes in which 7-ethoxycoumarin O-deethylase was used as an index activity. 9. It seems that for a principally CYP-metabolized substance such as CBZ, all liver-derived systems provide useful information for prediction of metabolic routes, rates and interactions.

Anticonvulsants↗

PubMed2001

Evaluation of 7-benzyloxy-4-trifluoromethylcoumarin, some other 7-hydroxy-4-trifluoromethylcoumarin derivatives and 7-benzyloxyquinoline as fluorescent substrates for rat hepatic cytochrome P450 enzymes.

1. The aim of this study was to evaluate a number of derivatives of 7-hydroxy-4-trifluoromethylcoumarin (HFC) and 7-benzyloxyquinoline (7BQ) as novel fluorescent substrates for monitoring rat hepatic cytochrome P450 (CYP) enzyme specificity in a 96- well plate format. The HFC derivatives examined comprised 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC), 3,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BTBFC), 2-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (2TFBFC), 3-(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (3TFBFC) and 3-(trifluoromethoxy)-7-benzyloxy-4-trifluoromethylcoumarin (3TFMeOBFC). 2. The CYP specificity of the fluorescent probe substrates was examined using characterized liver microsomes from male Sprague-Dawley rats treated with beta naphthoflavone (BNF), sodium phenobarbitone (NaPB), isoniazid, pregnenolone-16alpha-carbonitrile (PCN), dexamethasone (DEX) and methylclofenapate to induce CYP1A, CYP2B, CYP2E, CYP3A, CYP3A and CYP4A forms, respectively. Studies were also performed with microsomes from baculovirus-infected insect cells containing rat cDNA-expressed CYP1A1, CYP1A2, CYP2B1, CYP3A1 and CYP3A2. 3. BFC metabolism was most markedly induced by BNF and NaPB, whereas BFBFC metabolism was most markedly induced by PCN and DEX and BTBFC was not metabolized by rat liver microsomes. BFC was a high-affinity substrate for cDNA-expressed CYP1A1 and CYP2B1, whereas BFBFC exhibited a high affinity for CYP3A1 and CYP3A2. 4. The metabolism of 2TFBFC and 3TFBFC was induced by NaPB, PCN and DEX. 3TFBFC was a relatively specific substrate for cDNA-expressed CYP2B1, whereas 2TFBFC could be metabolized by CYP2B1, CYP3A1 and CYP3A2. 5. 3TFMeOBFC metabolism was markedly induced by BNF treatment and 3TFMeOBFC was extensively metabolized by cDNA-expressed CYP1A1. 6. The metabolism of 7BQ to 7-hydroxyquinoline was induced by treatment with PCN and DEX. 7BQ was a substrate for cDNA-expressed CYP3A2 and to a lesser extent for CYP3A1. 7. In summary, some of the HFC derivatives studied and 7BQ are useful fluorescent probe substrates for rat CYP enzymes. BFC appears to be a probe for CYP1A and CYP2B, 2TFBFC for CYP2B and CYP3A and 3TFBFC for CYP2B. While 3TFMeOBFC appears to be a relatively specific probe for CYP1A1, both BFBFC and 7BQ are good probes for the induction of CYP3A.

Animals↗

PubMed2001

Quantitative structure-activity relationships (QSARs) within substrates of human cytochromes P450 involved in drug metabolism.

The results of quantitative structure-activity relationship (QSAR) analyses are reported for structurally diverse series of chemicals which act as substrates or inhibitors for human hepatic microsomal cytochromes P450 (CYP). In particular, this study focuses on the major catalysts of drug metabolism in man, namely CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. It is found that good correlations (with correlation coefficients ranging from R = 0.94 to 0.99) with P450 binding affinity (Km and K(D)) or competitive inhibition (Ki) values are obtained in each case, especially when consideration of hydrogen bonding parameters are included in the QSAR analysis, together with the number of pi-pi stacking interactions.

Cytochrome P-450 Enzyme System↗

PubMed2001

An assessment of human liver-derived in vitro systems to predict the in vivo metabolism and clearance of almokalant.

The ability of various human derived in vitro systems to predict various aspects of the in vivo metabolism and kinetics of almokalant have been investigated in a multicenter collaborative study. Although almokalant has been withdrawn from further clinical development, its metabolic and pharmacokinetic properties have been well characterized. Studies with precision-cut liver slices, primary hepatocyte cultures, and hepatic microsomal fractions fortified with UDP-glucuronic acid all suggested that almokalant is mainly glucuronidated to the stereoisomers M18a and M18b, which is in good agreement with the results in vivo. Both in vivo and in vitro studies indicate that the formation of M18b dominates over that of M18a, although the difference is more pronounced with the in vitro systems. Molecular modeling, cDNA-expressed enzyme analysis, correlation analysis, and inhibition studies did not clearly indicate which P450 enzymes catalyze the oxidative pathways, which may indicate a problem in identifying responsible enzymes for minor metabolic routes by in vitro methods. All of the in vitro systems underpredicted the metabolic clearance of almokalant, which has previously been reported to be a general problem for drugs that are cleared by P450-dependent metabolism. Although few studies on in vivo prediction of primarily glucuronidated drugs have appeared, in vitro models may consistently underpredict in vivo metabolic clearance. We conclude that in vitro systems, which monitor phase II metabolism, would be beneficial for prediction of the in vivo metabolism, although all of the candidate liver-derived systems studied here, within their intrinsic limitations, provided useful information for predicting metabolic routes and rates.

Anti-Arrhythmia Agents↗

PubMed2000

Interactions between redox partners in various cytochrome P450 systems: functional and structural aspects.

The various types of redox partner interactions employed in cytochrome P450 systems are described. The similarities and differences between the redox components in the major categories of P450 systems present in bacteria, mitochondria and microsomes are discussed in the light of the accumulated evidence from X-ray crystallographic and NMR spectroscopic determinations. Molecular modeling of the interactions between the redox components in various P450 mono-oxygenase systems is proposed on the basis of structural and mutagenesis information, together with experimental findings based on chemical modification of key residues likely to be associated with complementary binding sites on certain typical P450 isoforms and their respective redox partners.

Animals↗

PubMed2000

Quantitative structure-activity relationships in a series of endogenous and synthetic steroids exhibiting induction of CYP3A activity and hepatomegaly associated with increased DNA synthesis.

The results of a quantitative structure-activity relationship (QSAR) study on a total of 14 steroids exhibiting induction of a CYP3A-associated activity and increase in liver weight/DNA synthesis is reported. It is found that different, but related, structural descriptors correlate with increase in ethylmorphine N-demethylase activity (r=0.92) and with the increase in liver weight (r=0.78) and DNA synthesis (r=0.78). Although there is a strong correlation between increase in liver weight and DNA content (r=0.999), neither of these correlated with ethylmorphine N-demethylase activity. These findings are discussed in the light of CYP3A induction, substrate specificity and inhibition; a proposed model of human CYP3A4 based on sequence homology with CYP102, a bacterial P450 of known crystal structure, demonstrates the possible mode of interaction between substrates and inhibitors within the putative active site.

Aryl Hydrocarbon Hydroxylases↗

PubMed2000

On the recognition of mammalian microsomal cytochrome P450 substrates and their characteristics: towards the prediction of human p450 substrate specificity and metabolism.

The characteristics of mammalian microsomal P450 xenobiotic substrates are described, particularly with reference to the major P450 isoforms associated with drug metabolism in humans. It is further reported that a relatively small number of molecular, electronic, and physico-chemical properties are required to discriminate between chemicals that exhibit specificity for human P450 isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Molecular templates of superimposed substrates are shown to be complementary with the putative active sites of the relevant enzymes, thus enabling a possible prediction of P450 specificity from structure. Factors contributing to metabolic clearance and binding affinity are also discussed, and methods for their calculation are described.

Animals↗

PubMed2000

Structural characteristics of human P450s involved in drug metabolism: QSARs and lipophilicity profiles.

The factors with human P450 substrate selectivity are reviewed, together with extensive tabulation of quantitative relationships between structure and activity for many species of P450 substrates, inducers and inhibitors. In addition, the physicochemical characteristics (logP and pK(a) values) of heptic microsomal P450 substrates are collated, such that comparisons can be made on the grounds of compound lipophilicities.

Animals↗

PubMed2000

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette.

A capillary array electrophoresis DNA sequencer is reported based on a micromachined sheath-flow cuvette as the detection chamber. This cuvette is equipped with a set of micromachined features that hold the capillaries in precise registration to ensure uniform spacing between the capillaries, in order to generate uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes, which operate in the analog mode. A high-gain transimpedance amplifier is used for each photodiode, providing high duty-cycle detection of fluorescence.

DNA↗

PubMed2000

Placental transfusion: umbilical cord clamping and preterm infants.

OBJECTIVE: To investigate the clinical effects of early versus late cord clamping in preterm infants. STUDY DESIGN: A total of 32 premature infants were prospectively randomized. The following parameters were measured: Initial spun hematocrit (Hct), hemoglobin (Hgb), red blood cell (RBC) counts, frequency of blood transfusions, peak serum bilirubin, mean blood pressure (MBP), oxygen index, intraventricular hemorrhage, and significant patent ductus arteriosus (PDA). RESULTS: Over the 4-week study period, the delayed cord clamping (DCC) group exhibited a decrease in the frequency of blood transfusion (p < 0.001) and also a decrease in albumin transfusions over the first 24 hours (p < 0.03). MBP in the first 4 hours was higher in the DCC group (p < 0.01), and there were statistically significant increases in Hct (21%), Hgb (23%), and RBC count (21%) compared with the early cord clamping group. The risks of patent ductus arteriosus, hyperbilirubinemia, or intraventricular hemorrhage were similar in both groups. Late clamping of the umbilical cord had little or no effect on the oxygen index. CONCLUSION: DCC significantly reduced the requirement for blood and albumin transfusion. It also increased the initial Hct, RBC count, Hgb levels, and MBP.

Blood Component Transfusion↗

PubMed2000

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform.

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.

Amino Acid Sequence↗