Biomedical subjects

S W Grimm

Publications and source records attributed to S W Grimm.

7 recordsLinked to original sources

PubMed2002

Reversible and irreversible inhibition of CYP3A enzymes by tamoxifen and metabolites.

1. Preliminary studies have identified cytochrome P450 (CYP) 3A4 and CYP1B1 as the human CYPs inhibited by tamoxifen. To quantify the inhibitory potency of tamoxifen and its major metabolites, the metabolism of three substrates of CYP3A, midazolam, diltiazem and testosterone, and 7-ethoxyresorufin as a substrate of CYP1B1 were examined in catalytic assays carried out using human liver microsomes and cDNA-expression systems. 2. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen reversibly inhibited midazolam 1'-hydroxylation, diltiazem N-demethylation and testosterone 6beta-hydroxylation with K(i) ranging from 3 to 37 micro M in human liver microsomes. Tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and 3-hydroxytamoxifen also reversibly inhibited the activity of cDNA-expressed CYP3A4, CYP3A5 and CYP1B1. 3. Tamoxifen and N-desmethyltamoxifen exhibited time-dependent inactivation of testosterone 6beta-hydroxylation by cDNA-expressed CYP3A4 (+ cytochrome b5) yielding k(inact) and K(i) of 0.04 min(-1) and 0.2 micro M for tamoxifen and 0.08 min(-1) and 2.6 micro M for N-desmethyltamoxifen. A metabolic intermediate complex (MIC) was also formed by tamoxifen and N-desmethyltamoxifen with CYP3A4 (+ cytochrome b5) and CYP3A4 but not with CYP3A5 or CYP3A7. Pre-incubation with 4-hydroxytamoxifen and 3-hydroxytamoxifen did not result in any CYP3A inactivation or detectable MIC formation. There was no detectable time-dependent inactivation or MIC formation with tamoxifen or metabolites with CYP1B1. 4. These data indicate that tamoxifen and its three major metabolites are effective inhibitors of CYP3A in vitro and that tamoxifen and N-desmethyltamoxifen are effective mechanism-based inhibitors. Thus, caution should be exercised when tamoxifen is coadministered with other CYP3A substrates.

Antineoplastic Agents, Hormonal↗

PubMed1997

Inhibition of human drug metabolizing cytochromes P450 by anastrozole, a potent and selective inhibitor of aromatase.

Anastrozole (2,2'[5(1H-1,2,4-triazol-1-ylmethyl)-1,3-phenylene]- bis(2-methylproprionitrile)) is a potent third-generation inhibitor of aromatase, currently marketed as a treatment for postmenopausal women with advanced breast cancer. While its potency and selectivity for inhibition of estrogen synthesis has been established in both preclinical and clinical studies, this study used in vitro methods to examine the effects of anastrozole on several drug metabolizing CYP enzymes found in human liver. Human liver microsomes were co-incubated with anastrozole and probe substrates for CYP1A2 (phenacetin), CYP2A6 (coumarin), CYP2C9 (tolbutamide), CYP2D6 (dextromethorphan), and CYP3A (nifedipine). The formation of the CYP-specific metabolites following co-incubation with various anastrozole concentrations was determined to establish IC50 and Ki values for these enzymes. While anastrozole did not inhibit CYP2A6 and CYP2D6 activities at concentrations below 500 microM, this compound inhibited CYP1A2, CYP2C9, and CYP3A activities with Ki values of 8, 10, and 10 microM, respectively. Dixon plots used to determine the Ki values for the inhibition of CYP1A2 and CYP3A activities by anastrozole were biphasic, indicating additional lower affinity Ki values. Major metabolites of anastrozole did not retain the ability to inhibit the metabolism of nifedipine (CYP3A). The results of this study indicate that, although anastrozole can inhibit CYP1A2, 2C9, and 3A-mediated catalytic activities, this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at physiologically relevant concentrations achieved during therapy with Arimidex (Zeneca, Ltd., Macclesfield, UK) 1-mg.

Anastrozole↗

PubMed1995

Selectivity and kinetics of inactivation of rabbit hepatic cytochromes P450 2B4 and 2B5 by N-aralkylated derivatives of 1-aminobenzotriazole.

The kinetics of mechanism-based inactivation of phenobarbital-inducible rabbit hepatic cytochromes P450 2B4 and 2B5 by N-benzyl-(BBT) and N-alpha-methylbenzyl (alpha MB) 1-aminobenzotriazole were investigated using reconstituted P450 2B4, a stable heterologous expression system, and hepatic microsomes. Low micromolar concentrations of the 1-aminobenzotriazole derivatives caused reversible inhibition as well as rapid inactivation of reconstituted P450 2B4 and recombinant P450 2B4 and 2B5. In contrast, even at a 1000-fold higher concentration, aminobenzotriazole inactivated the expressed P450 2B enzymes less rapidly. Preincubation of phenobarbital-induced hepatic microsomes with BBT and alpha MB resulted in concentration-dependent decreases in marker activities of P450 2B4 and 2B5, benzyloxyresorufin O-debenzylase and androstenedione 15 alpha-hydroxylase, respectively. BBT caused the inactivation of P450 2B4 and 2B5 in hepatic microsomes with apparent Kl values of 1.9 and 2.4 microM and maximal rate constants of 0.29 and 0.18 min-1, respectively. alpha MB inactivated both P450 2B enzymes with similar Kl values (approximately 7 microM) and maximal rate constants only slightly higher for 2B4 compared with 2B5 (0.68 vs. 0.55 min-1). Similar P450 2B selectivity of BBT and alpha MB in both hepatic microsomes and the stable expression system further validates this new expression system and the use of the selective markers identified for 2B4 and 2B5 in hepatic microsomes. The results also provide a mechanistic basis for the high potency of the N-aralkylated 1-aminobenzotriazole derivatives in vivo and suggest that treatments that inactivate 2B4 will also lead to 2B5 inactivation.

Alkylation↗

PubMed1994

Catalytic selectivity and mechanism-based inactivation of stably expressed and hepatic cytochromes P450 2B4 and 2B5: implications of the cytochrome P450 2B5 polymorphism.

Cytochrome P450 (P450) 2B5 was recently found to be functionally distinct from three other rabbit P450 2B forms, based on androstenedione hydroxylase activities. In this investigation, we examined the frequency of the P450 2B5-null phenotype and the functional consequences of polymorphic P450 2B5 expression in hepatic microsomes from phenobarbital-treated rabbits. Four of the 10 animals examined did not have detectable levels of P450 2B5 mRNA and exhibited much lower microsomal androstenedione 15 alpha- and 16 alpha-hydroxylase activities. The 15 alpha-hydroxylase activity was found to correlate (r = 0.91) with liver P450 2B5 mRNA. P450 2B4 and 2B5 were stably expressed in human kidney 293 cells to further characterize substrate specificities and to investigate mechanism-based inactivation by phencyclidine. P450 2B4 was 4-16-fold more active than 2B5 towards benzphetamine, 7-ethoxycoumarin, methylenedioxybenzene, and pentoxyresorufin. Benzyloxyresorufin O-debenzylase activity was 160-fold higher for P450 2B4 than P450 2B5. Anti-P450 2B4 IgG inhibited benzyloxyresorufin O-debenzylation nearly completely in untreated and phenobarbital-induced liver microsomes. Phencyclidine selectively inactivated P450 2B4, compared with 2B5, in both human kidney 293 cell and liver microsomes. Poor inactivation of P450 2B5 by phencyclidine was found to be a result of its low maximal rate constant. Results of this study establish the idea that the metabolic consequences of phenobarbital induction depend on the potential of animals to express functionally variant P450 2B forms. Furthermore, we conclude that one or more of the 11 amino acid differences between these highly related P450 forms are critical to their substrate specificities and selective inactivation.

Androstenedione↗

PubMed1993

Cloning, sequencing, and functional studies of phenobarbital-inducible forms of cytochrome P450 2B and 4B expressed in rabbit kidney.

Expression of several forms of cytochrome P450 2B and of P450 4B1 in rabbit kidney was investigated by cloning from cDNA libraries constructed with renal mRNA from animals treated with phenobarbital. Isolation and sequencing of several cDNAs demonstrated that: (i) cytochrome P450 2B-B0 can be found in rabbit kidney along with a newly discovered form of P450 2B termed "P450 2B-Bx." P450 2B-Bx differs from P450 2B-B0 at 25 nucleotide positions and at four positions in the derived sequence of 491 amino acids. Two previously identified forms of cytochrome P450 2B, 2B-B1 and 2B-B2, are not detected in rabbit kidney. cDNA encoding cytochrome P450 4B1 was also cloned from the kidney library and found to be identical in sequence to cDNAs cloned from rabbit hepatic and pulmonary libraries. Analysis of renal mRNA indicates that forms of cytochrome P450 2B and P450 4B1 are expressed in a number of species but induced by phenobarbital in rabbit only (4B1) or rabbit and hamster (2B). Relatively high levels of mRNA related to P450 4B1 were detected in samples from untreated and phenobarbital-treated mice. Analysis of protein by immunoblotting was less sensitive but produced results consistent with those obtained by analysis of mRNA; protein related to cytochrome P450 2B was detected in renal microsomal samples from rabbit and hamster (phenobarbital > untreated), and protein related to P450 4B1 in samples from rabbits (phenobarbital > untreated) and mice (phenobarbital = untreated). The four forms of cytochrome P450 2B were expressed in COS-7 cells, and their activities were evaluated with androstenedione, testosterone, and 7-ethoxycoumarin as substrates. Three of the P450 2B forms, B0, B1, and Bx, metabolize these substrates in a manner characterized by preference for 16 beta-hydroxylation of androstenedione, low testosterone 16-hydroxylation, and high ethoxycoumarin O-deethylation. The fourth form, P450 2B-B2, is catalytically distinct from the others, with activities characterized by high androstenedione 16 alpha- and 15 alpha-hydroxylation and high testosterone 16-hydroxylation. Since P450 2B-B2 is catalytically distinct from the other forms, the metabolic profiles of phenotypes that include P450 2B-B2 might differ significantly from those of phenotypes that lack P450 2B-B2.

Amino Acid Sequence↗

PubMed1993

Hybrid enzymes for structure-function analysis of cytochrome P-450 2B11.

Previous work has shown that P-450 2B11 is responsible for the unique ability of dogs to metabolize and eliminate certain highly-chlorinated biphenyls such as 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), whereas the related P-450 2B forms in rat and rabbit are unable to metabolize the compound to any significant degree. To determine the structural basis for this functional diversity, hybrid enzymes were generated. Success with this approach required a careful choice of second enzyme and common substrate with which to assess the functional integrity of the hybrid proteins. The choices of P-450 2B5 from rabbit as the second enzyme and androstenedione as the substrate were based in part on the finding that P-450 2B11 and P-450 2B5 hydroxylate androstenedione with similar overall activities but distinct profiles. Enzymatic studies with eight hybrid enzymes provided evidence for two regions of P-450 2B11 and 2B5, between residues 95-239 and 240-370, that appear to be involved in defining substrate specificity for androstenedione, and three regions of P-450 2B11, between residues 95-239, 240-370, and 371-494, that contain amino acids necessary for metabolism of 245-HCB. This deliberate approach to the creation of hybrid cytochromes P-450 has generated a series of enzymes that will be central to further structure-function studies of the cytochromes P-450 2B.

Androstenedione↗

PubMed1991

Molecular basis for a functionally unique cytochrome P450IIB1 variant.

Liver microsomes from phenobarbital-treated rats of four inbred strains expressing distinct allelic variants of cytochrome P450IIB1 were analyzed. The Wistar Munich (WM) strain exhibited 5- to 10-fold lower androstenedione 16 beta-hydroxylase activity (a specific P450IIB1 marker) than the Lewis, Wistar Kyoto, and Wistar Furth strains. The androstenedione 16 beta-hydroxylase in the WM liver microsomes was refractory to inactivation by N-(2-p-nitrophenethyl)chlorofluoroacetamide, a selective P450IIB1 inactivator in the other three strains. Purified P450IIB1-WM was insensitive to the inactivator and exhibited 5-fold lower androstenedione 16 beta-hydroxylase, testosterone 16-hydroxylase, and 7-ethoxycoumarin deethylase activities but the same benzphetamine demethylase activity and slightly higher androstenedione 16 alpha-hydroxylase activity than a P450IIB1 purified from outbred Sprague-Dawley rats, which appears to correspond to the form in Lewis rats. The stereoselectivity of androstenedione 16-hydroxylation catalyzed by P450IIB1-WM (16 beta-OH:16 alpha-OH = 1.4) is thus distinct from that (16 beta-OH:16 alpha-OH = 12-15) of other P450IIB1 preparations described. A cDNA encoding P450IIB1-WM was cloned and sequenced, revealing a single amino acid substitution (Gly-478----Ala) compared with the published sequence (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797). Heterologous expression of P450IIB1 and P450IIB1-WM confirmed the striking difference in androstenedione metabolite profiles, strongly implicating the involvement of Ala-478 in defining the distinctive catalytic properties of P450IIB1-WM.

Alleles↗