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L V Favreau

Publications and source records attributed to L V Favreau.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of CYP3A4 in a rapid microtiter plate assay using recombinant enzyme and in human liver microsomes using conventional substrates.

Cytochrome P450 inhibition studies are performed in the pharmaceutical industry in the discovery stage to screen candidates that may have the potential for clinical drug-drug interactions. A 96-well microtiter plate assay using recombinant cytochrome P450 (Supersomes) has been used to increase the overall throughput. The IC(50) values for the inhibition of CYP3A4 by 52 new chemical entities (NCEs) were determined using the Supersomes assay with resorufin benzyl ether as a substrate, and the data were compared with those obtained in human liver microsomes (HLM) using midazolam as a substrate. Among the 52 compounds tested, 25 showed IC(50) values within a 5-fold difference in the two assays. For all compounds that showed a >5-fold difference, the IC(50) values in the Supersomes assay were lower than those obtained in HLM, except for one compound. Further studies suggested that this discrepancy was not related to difference in protein concentrations between the two assays. In addition, the IC(50) values for 16 compounds with a wide range of inhibition potency were determined in HLM using testosterone and dextromethorphan as substrates. The results showed an 80 to 93% match within a 5-fold difference between the three probe substrates. However, for certain compounds including ketoconazole, there were substrate-dependent differences in the inhibition. The results suggest that the difference between the Supersomes and HLM could be partially attributed to differences in the substrate used, and to metabolism by other cytochrome P450s present in the HLM but not in the Supersomes. Furthermore, multiple CYP3A4 substrates should be used to improve the reliability of estimating potential drug-drug interaction of NCEs.

Cytochrome P-450 CYP3A↗
PubMed2001

Mechanism-based inactivation of CYP2D6 by 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine.

SCH 66712 [5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine] caused a time- and NADPH-dependent loss of CYP2D6 activity. The inactivation of human liver (HL) microsomal dextromethorphan O-demethylase activity, a prototype marker for CYP2D6, was characterized by a K(I) of 4.8 microM and a maximal rate constant of inactivation (k(inact)) of 0.14 min(-1). The inactivation of the recombinant CYP2D6 in Supersomes (r-CYP2D6) was characterized by a K(I) of 0.55 microM and a k(inact) of 0.32 min(-1). Extensive dialysis of the SCH 66712-inhibited enzyme failed to restore the activity to control levels (dialyzed reaction mixture lacking SCH 66712) for both HL microsomes and r-CYP2D6. Addition of glutathione, superoxide dismutase, or mannitol to the reaction mixture failed to protect CYP2D6 against SCH 66712-NADPH-catalyzed inactivation. Addition of quinidine, a reversible inhibitor of CYP2D6, to a preincubation mixture consisting of SCH 66712, HL microsomes, or Supersomes and NADPH partially protected CYP2D6 from inactivation. SCH 66712 also inhibited HL microsomal CYP3A4, CYP2C9, and CYP2C19; however, the concentrations required to inhibit those isoforms were 5- to 10-fold higher than those required to inhibit CYP2D6. These results demonstrate that SCH 66712 is a potent and fairly selective mechanism-based inhibitor of CYP2D6.

Chromatography, High Pressure Liquid↗
PubMed1999

Improved reliability of the rapid microtiter plate assay using recombinant enzyme in predicting CYP2D6 inhibition in human liver microsomes.

A higher throughput method of screening for the inhibition of recombinant CYP2D6 using a microtiter plate (MTP) assay was evaluated using 62 new chemical entities and compared to data from the dextromethorphan O-demethylase assay in human liver microsomes (HLM). The IC50 values for the two assays closely matched for 53 compounds (85%). Six of the variant nine compounds had higher IC50 values with the recombinant enzyme, whereas three had lower IC50 values with the recombinant enzyme. When the inhibition with the recombinant enzyme was determined at various time points, the IC50 values increased as the duration of the incubation increased for the six compounds with higher IC50 values in the MTP assay. The IC50 values at 10 min matched more closely the IC50 values in HLM (95% compared with 85%). For three compounds that showed comparable IC50 values in the two assays, and the three compounds with lower IC50 values in the MTP assay, the IC50 values did not change over time. These results suggest that the six compounds that showed higher IC50 values in the MTP assay at 45 min are substrates for CYP2D6. Using known CYP2D6 substrates, a similar phenomenon was observed, i.e., inhibition curves shifted to higher IC50 values as incubation time increased. These results indicate that the higher throughput MTP assay is more comparable to HLM if the IC50 values are determined at 10 min rather than the recommended 45 min. Furthermore, data acquisition at multiple time points may indicate if a compound is a potential substrate or metabolism/mechanism-based inhibitor for the enzyme.

Cytochrome P-450 CYP2D6↗
PubMed1995

The rat quinone reductase antioxidant response element. Identification of the nucleotide sequence required for basal and inducible activity and detection of antioxidant response element-binding proteins in hepatoma and non-hepatoma cell lines.

The antioxidant response element (ARE) found in the 5'-flanking region of the rat quinone reductase gene has been further characterized by mutational and deletion analysis. The results indicate that the 31-base pair ARE, which contains a 13-base pair palindromic sequence, can be further separated into three regions, all three of which are required for elevated basal level gene expression. These three regions include the proximal and distal half-sites as well as a 3'-flanking region consisting of 4 adenine nucleotides. Neither the proximal nor the distal half-site alone mediates transcriptional activation by beta-naphthoflavone. However, when placed together the two half-sites restore responsiveness to the inducer. Interestingly, the presence of only 1 of the 4 adenine nucleotides in the 3'-flanking region of the proximal half-site is required for responsiveness to the inducer. Point mutations within the ARE indicate that several nucleotides in both the proximal and distal half-sites are required for basal level gene expression. Electrophoretic mobility shift analysis using the ARE as the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from human Hep G2 cells. Further, this complex can be detected in nuclear extracts from rat liver and rat hepatoma cells but not in mouse Hepa 1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can also be detected in F9 cells which lack significant levels of Jun/Fos proteins. Although the rat ARE resembles the human quinone reductase ARE which contains a consensus TRE, the 2-nucleotide change in the core sequence (TGACTCA versus TGACTTG) eliminates the high affinity TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in Hep G2 and other cells with different properties than AP-1.

Animals↗
PubMed1993

Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Characterization of a DNA-protein interaction at the antioxidant responsive element and induction by 12-O-tetradecanoylphorbol 13-acetate.

We have previously identified a novel xenobiotic responsive element, which has been termed the antioxidant responsive element (ARE), in the 5'-flanking region of the rat quinone reductase gene (Favreau, L. V., and Pickett, C. B. (1991) J. Biol. Chem. 266, 4556-4561). This element is responsible for basal level expression of the gene as well as transcriptional activation by phenolic antioxidants and metabolizable planar aromatic compounds. In this communication, we demonstrate that hydrogen peroxide can act as an inducer through the ARE sequence, a phenomenon recently demonstrated for the glutathione S-transferase Ya subunit gene (Rushmore, T. H., Morton, M. R., and Pickett, C. B. (1991) J. Biol. Chem. 266, 11632-11639). To further characterize the quinone reductase ARE, we demonstrate by DNase I footprinting that in crude Hep G2 nuclear extracts a trans-acting factor exists which interacts with a region of DNA found within the 31-nucleotide ARE sequence. Furthermore, electrophoretic mobility shift assays demonstrate the presence of a specific DNA-protein complex which can be competed only by double-stranded oligonucleotides containing the ARE sequences from the quinone reductase and glutathione S-transferase Ya subunit genes. Methylation interference and protection assays indicate that several guanine residues found in the sequence GTGACTTGGC are involved in the binding of the nuclear factor(s) to the DNA. Although electrophoretic mobility shift assays indicate that the rat quinone reductase ARE does not contain a high affinity recognition site for in vitro translated c-Jun and c-Fos, 12-O-tetradecanoylphorbol 13-acetate can act as an inducer through the ARE sequence in Hep G2 cells.

Animals↗
PubMed1991

Transcriptional regulation of the rat NAD(P)H:quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants.

We have identified two regions in the 5'-flanking sequence of the rat quinone reductase gene that contain xenobiotic responsive elements. The DNA sequence of the first region spans nucleotides -393 to -352 of the 5'-flanking region and shares sequence identity with the xenobiotic responsive element (XRE) described for the cytochrome P-450 CYPIA1 gene. The DNA sequence of the second region spans nucleotides -434 to -404 of the 5'-flanking region of the quinone reductase structural gene. When a synthetic oligonucleotide corresponding to nucleotides -434 to -404 was inserted in front of a heterologous promoter linked to the chloramphenicol acetyltransferase structural gene, an increase in basal level expression as well as responsiveness to beta-naphthoflavone and t-butylhydroquinone, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, was observed. The sequence, -434 to -404, did not have any sequence identity with the XRE but shared a large degree of identity with the antioxidant responsive element recently described for the rat glutathione S-transferase Ya subunit gene (Rushmore, T. H., King, R. G., Paulson, K. E., and Pickett, C. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3826-3830; Rushmore, T. H., and Pickett, C. B. (1990) J. Biol. Chem. 265, 14648-14653). These results indicate that the antioxidant responsive element can be distinguished functionally from the classical XRE and is also involved in the regulation of the quinone reductase gene by planar aromatic compounds and phenolic antioxidants.

Amino Acid Sequence↗
PubMed1989

Rat liver NAD(P)H: Quinone reductase. Regulation of quinone reductase gene expression by planar aromatic compounds and determination of the exon structure of the quinone reductase structural gene.

We have determined the effect of beta-naphthoflavone and the azo dye, sudan III, on the level of quinone reductase mRNA in a responsive rat hepatoma cell line. Our data indicate that both of these planar aromatic compounds produce a 4-5-fold elevation in quinone reductase mRNA. The induction of quinone reductase mRNA can be blocked by cycloheximide, suggesting a requirement for ongoing protein synthesis in the induction process. We have determined the exon structure of the quinone reductase structural gene. The gene is separated into six exons by five introns. A "TATA" box is located 29 base pairs upstream from the transcription initiation site. A "CCAAT" sequence is found at position -129, and an inverted "GC" box is located at position -78. Quinone reductase promoter-chlor-amphenicol acetyltransferase fusion genes containing different lengths of the 5'-flanking region were transfected into rat and human hepatoma cells. Treatment of the transfected cells with beta-naphthoflavone or sudan III resulted in a 4-5-fold elevation in chloramphenicol acetyltransferase activity. These data suggest the presence of a cis-acting regulatory element(s) in the 5'-flanking region of the quinone reductase structural gene which regulates inducible expression.

Amino Acid Sequence↗
PubMed1988

Further characterization of RLM2 and comparison with a related form of cytochrome P-450, RLM2b.

We have extended the characterization of RLM2, a constitutive form of rat liver cytochrome P-450, using immunochemical means to quantitate its presence in microsomes, to follow its development in maturing male and female rats, and to determine its response to prototypical P-450 inducers. In addition, RLM2 is compared to RLM2b, a form of P-450 with similar migration on SDS-PAGE and NH2-terminal amino acid sequence. RLM2b is expressed in both sexes at a level of 0.08 nmol/mg microsomal protein at 2 weeks of age. In female rats, this level is unchanged with maturation. However, in the male, the level declined with maturation to reach 0.02 nmol/mg protein by 12 weeks of age. RLM2 is a male-specific form of cytochrome P-450. Originally absent in the 2-week-old rat, it reached a level of 0.03 nmol/mg protein in the adult male, its appearance and increase coinciding with the onset of puberty. Both phenobarbital and 3-methylcholanthrene induced microsomal levels of RLM2b in the adult male and female rat. RLM2, however, was suppressed in the male rat, 58 and 42%, respectively, by the same treatments. RLM2b and RLM2 each catalyze a unique spectrum of hydroxytestosterone metabolites. RLM2b is highly site specific. In contrast, RLM2 produces several isomeric products in the same region of the testosterone molecule. Substitution of the acetyl group of progesterone for the 17-hydroxy group of testosterone did not alter the site specificity of RLM2b, but did alter it for RLM2, indicating, further, a difference in the active site conformation of the two enzymes. Although RLM2b and RLM2 responded differently to inducers and to a changing physiology during maturation, and were functionally quite distinct, the proteins showed a high degree of immunologic relatedness which is suggestive of significant structural similarities. Structural differences do exist, however, as alpha-chymotryptic digestion formed a number of peptide fragments that differed between the two proteins.

Amino Acid Sequence↗
PubMed1988

Cytochrome P-450 alterations in the BB/Wor spontaneously diabetic rat.

The hepatic content of two individual cytochrome P-450 enzymes was analyzed in spontaneously diabetic (BB/Wor) male rats. The major male-specific form, RLM5, was found to be slightly decreased in livers of male rats shortly after the onset of diabetes. In contrast, the level of RLM6 was elevated in livers of diabetic rats that had not received insulin and had become ketotic. These results confirm that the changes in some individual forms of cytochrome P-450 after chemical (streptozotocin) induction of diabetes are also seen in the spontaneously diabetic animal. The earlier observed alterations in cytochrome P-450 are therefore due to the state of diabetes and not to inductive or depressive effects of streptozotocin.

Animals↗
PubMed1988

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: formation of a novel metabolite by a female-specific P-450.

Previous studies demonstrated that liver microsomes from untreated rats catalyze the omega, omega-1, and omega-2 hydroxylation of prostaglandins [K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489]. The current study examined the regioselectivity of hydroxylation of PGE1 and PGE2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH-P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE1, RLM 5 catalyzed solely the omega-1 hydroxylation and 5a catalyzed primarily the omega-1 and little omega and omega-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE1 and PGE2 at the omega-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is omega-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE1 hydroxylation demonstrated that the affinity of f4 for PGE1 is approximately 100-fold higher than that of RLM 5; the Km values for f4, monitoring 19- and 17-hydroxylation of PGE1, were about 10 microM. Surprisingly, cytochrome b5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE2 by RLM 5 was at the omega, omega-1, and omega-2 sites, demonstrating a lesser regioselectivity than with PGE1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b5 requirement.

Animals↗
PubMed1988

Composition changes in hepatic microsomal cytochrome P-450 during onset of streptozocin-induced diabetes and during insulin treatment.

Monospecific polyclonal antibodies to five constitutive hepatic microsomal cytochromes P-450 were prepared. These antibodies were used to monitor alterations in the content of the enzymes in livers of diabetic male rats. Within 3 wk of onset of streptozocin-induced diabetes, immunodetectable levels of RLM3 and RLM5 were decreased by 85 and greater than 95%, respectively. Insulin treatment for 1 wk reversed the decline in these isozymes and restored RLM3 to 60% and RLM5 to 53% of levels found in the untreated rat. After a 2nd wk of therapy, these levels were returned to 86 and 92%, respectively. In contrast, the levels of RLM5b and RLM6 were elevated in diabetes 1.7- and 8-fold, respectively. Insulin treatment for 1 wk only slightly decreased the levels of RLM5b but completely reduced RLM6 levels to those seen in age-matched untreated rats. After the 2nd wk of insulin treatment, the level of RLM5b was almost completely restored to normal, with no additional change in the RLM6 level. The level of a fifth enzyme, RLM5a, was not markedly altered by diabetes or by insulin treatment. The results suggest there are at least three types of responses by constituents of the cytochrome P-450 population to diabetes: no change in the microsomal content, a rapid increase when insulin level declines and restoration when insulin is supplied, and a rapid decline when insulin level declines and a restoration by insulin treatment.

Animals↗
PubMed1987

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes.

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.

Amino Acid Sequence↗
PubMed1987

Decrease in the levels of a constitutive cytochrome P-450 (RLM5) in hepatic microsomes of diabetic rats.

Cytochrome P-450 dependent hydroxylation of testosterone has been measured in hepatic microsomes of control, diabetic and insulin-treated diabetic rats. The observed decrease in testosterone 16 alpha-hydroxylase activity in diabetes, an activity previously shown to be largely due to RLM5, was accompanied by a dramatic decrease in immunodetectable RLM5. Diabetic rats which received insulin had elevated testosterone 16 alpha-hydroxylase activity relative to the diabetic animals, which was accompanied by a corresponding increase in the levels of RLM5. These results provide evidence that specific constitutive cytochrome P-450 enzymes are altered in the diabetic state and that these changes are not permanent since they can be overcome, at least partially, by insulin replacement therapy.

Animals↗
PubMed1987

Biochemical and immunological identity of the hepatic peroxisomal and microsomal trans-2-enoyl CoA hydratase bifunctional protein.

In the present study, the hepatic microsomal and peroxisomal bifunctional trans-2-enoyl CoA hydratases were isolated and purified from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These two enzymes (microsomal and peroxisomal) were purified with the identical purification procedures and had identical molecular masses of 76 kDa. A single band was observed on an electrophoretic gel of an equimixture of the two proteins. Both preparations had identical pI's of 8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5 for the hydratase activity. Two-dimensional gel analysis of an equimixture of the two preparations showed only one band. Ouchterlony double-diffusion analysis showed that an antibody raised against the purified microsomal enzyme interacted at a point with the peroxisomal enzyme, indicating immunologic identity. Western blot analysis demonstrated that the antibody formed a single band with total microsomal and peroxisomal fractions. The antibody inhibited the enzymatic activities of both preparations in a similar manner. Interestingly, the antibody had a markedly greater inhibitory effect on the reductase activity of the two enzyme preparations, and a much less inhibitory effect on the hydratase activity, suggesting that the antigenic determinants reside at or near the catalytic site of the reductase portion of the protein. These results suggest that the microsomal and peroxisomal bifunctional proteins are identical.

Animals↗
PubMed1987

Fingerprinting rat liver microsomal cytochromes P-450 as a means of delineating sexually distinctive forms.

The cytochrome P-450 fraction of microsomes separated on lauric acid AH-Sepharose 4B columns contains about 75% of the microsomal P-450. This was fingerprinted by means of two dimensional isoelectric focusing/SDS-PAGE. Separation of the fraction by highly reproducible, standard procedures on carboxymethyl Sepharose CL6B into four fractions allowed ready isolation and purification of seven forms of P-450, RLM2, 2b, 3, f4, 5, 5a and f5a. Comparison of the four fractions CMI, CMII, CMIII and CMIV revealed qualitative differences in the proteins contained in CMI and CMII of male and female rats. Identification of these proteins revealed RLM2, present in the CMI fraction of adult male rats, is not present in detectable levels in the comparable fraction from females. Similarly, RLM3 and 5 were present in the CMII fraction of male rats but could not be detected in the corresponding fraction of females. Instead, another protein, fRLM4, was found in the females. RLM5a, found in the CMII fraction of males, was also present in females. Examination of the physical properties of these P-450 proteins revealed those isolated in the CMI and CMII fractions to have fairly neutral isoelectric points (7.1-7.6). Based upon the NH2-terminal amino acid sequence, three classes of constitutive forms of P-450 can be recognized. All of the constitutive forms have methionine in position one and leucine in position seven. By comparing sequence homologies, RLM2 and 2b form one sub-class, RLM3, f4 and 5 form a second sub-class, and P-450f and RLM5a form a third sub-class.

Animals↗
PubMed1986

Differences in the spectral interactions between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 enzymes.

The interaction between NADPH-cytochrome P-450 reductase and a series of cytochrome P-450 isozymes was investigated using UV-visible spectrophotometry. In the absence of substrate the interactions between the reductase and RLM3, RLM5, and RLM5a were tight, exhibiting sub-micromolar dissociation constants and resulted in type I spectra of varying magnitude from which the following increases in the proportion of high spin hemoprotein were calculated; RLM3 (7%), RLM5 (36%), RLM5a (6%), LM2 (29%), RLM2 (0%). Preincubation of LM2 with its type I substrate benzphetamine increased the affinity of the cytochrome for the reductase. Using initial estimates of the P-450 spin states in the absence of reductase in conjunction with the spectral binding data and equations relating these parameters to the microequilibria for the association of reductase with high or low spin P-450, RLM3, RLM5, RLM5a and LM2 were shown to bind significantly more tightly to high spin P-450. The relevance of this data to the understanding of spin state influence on P-450 reduction is discussed.

Animals↗