scholarly journals Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16α-carbonitrile-inducible rat cytochromes P-450

1987 ◽  
Vol 248 (1) ◽  
pp. 301-304 ◽  
Author(s):  
T S Barnes ◽  
P M Shaw ◽  
M D Burke ◽  
W T Melvin
Keyword(s):  
Monoclonal Antibodies ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
The Other ◽  
Male Rat ◽  
Rat Liver Microsomes ◽  
Human Cytochrome ◽  
Male Specific ◽  
Cytochrome P 450

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.

Metabolism of rifabutin and its 25-desacetyl metabolite, LM565, by human liver microsomes and recombinant human cytochrome P-450 3A4: relevance to clinical interaction with fluconazole.

1997 ◽  
Vol 41 (5) ◽  
pp. 924-926 ◽  
Author(s):  
C B Trapnell ◽  
C Jamis-Dow ◽  
R W Klecker ◽  
J M Collins
Keyword(s):  
Human Liver ◽  
High Performance ◽  
Opportunistic Infections ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Hepatic Enzymes ◽  
Microsomal Enzymes ◽  
Human Cytochrome ◽  
Immunodeficiency Virus ◽  
Cytochrome P 450

Rifabutin and fluconazole are often given concomitantly as therapy to prevent opportunistic infections in individuals infected with the human immunodeficiency virus. Recent reports have shown increased levels of rifabutin and its 25-desacetyl metabolite, LM565, in plasma when rifabutin is administered with fluconazole. Since fluconazole is known to inhibit microsomal enzymes, this study was undertaken to determine if this rifabutin-fluconazole interaction was due to an inhibition of human hepatic enzymes. The metabolism of both rifabutin and LM565 was evaluated in human liver microsomes and recombinant human cytochrome P-450 (CYP) 3A4 in the presence of fluconazole and other probe drugs known to inhibit CYP groups 1A2, 2C9, 2D6, 2E1, and 3A. The concentrations of rifabutin (1 microg/ml), LM565 (1 microg/ml), and fluconazole (10 and 100 microg/ml) used were equal to those observed in plasma after the administration of rifabutin and fluconazole at clinically relevant doses. High-performance liquid chromatography was used to assess the metabolism of rifabutin and LM565. Rifabutin was readily metabolized to LM565 by human microsomes, but the reaction was independent of NADPH and was not affected by the P-450 inhibitors. No rifabutin metabolism by recombinant CYP 3A4 was found to occur. LM565 was also metabolized by human microsomes to two products, but metabolism was dependent on NADPH and was affected by certain P-450 inhibitors. In addition, LM565 was readily metabolized by the recombinant CYP 3A4 to the same two products found with its metabolism by human microsomes. Therefore, rifabutin is metabolized by human microsomes but not via cytochrome P-450 enzymes, whereas LM565 is metabolized by CYP 3A4.

A human cytochrome P-450 characterized by inhibition studies as the sparteine–debrisoquine monooxygenase

1984 ◽  
Vol 62 (7) ◽  
pp. 860-862 ◽  
Author(s):  
T. Inaba ◽  
M. Nakano ◽  
S. V. Otton ◽  
W. A. Mahon ◽  
W. Kalow
Keyword(s):  
Correlation Coefficient ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Human Cytochrome ◽  
Competitive Inhibitors ◽  
Inhibition Constants ◽  
Inhibition Studies ◽  
Cytochrome P 450

The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.

scholarly journals Nontargeted Metabolomics by High-Resolution Mass Spectrometry to Study the In Vitro Metabolism of a Dual Inverse Agonist of Estrogen-Related Receptors β and γ, DN203368

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 776
Author(s):  
Sin-Eun Kim ◽  
Seung-Bae Ji ◽  
Euihyeon Kim ◽  
Minseon Jeong ◽  
Jina Kim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Human Liver ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Rat Liver Microsomes ◽  
High Resolution Mass ◽  
Resolution Mass

DN203368 ((E)-3-[1-(4-[4-isopropylpiperazine-1-yl]phenyl) 3-methyl-2-phenylbut-1-en-1-yl] phenol) is a 4-hydroxy tamoxifen analog that is a dual inverse agonist of estrogen-related receptor β/γ (ERRβ/γ). ERRγ is an orphan nuclear receptor that plays an important role in development and homeostasis and holds potential as a novel therapeutic target in metabolic diseases such as diabetes mellitus, obesity, and cancer. ERRβ is also one of the orphan nuclear receptors critical for many biological processes, such as development. We investigated the in vitro metabolism of DN203368 by conventional and metabolomic approaches using high-resolution mass spectrometry. The compound (100 μM) was incubated with rat and human liver microsomes in the presence of NADPH. In the metabolomic approach, the m/z value and retention time information obtained from the sample and heat-inactivated control group were statistically evaluated using principal component analysis and orthogonal partial least-squares discriminant analysis. Significant features responsible for group separation were then identified using tandem mass spectra. Seven metabolites of DN203368 were identified in rat liver microsomes and the metabolic pathways include hydroxylation (M1-3), N-oxidation (M4), N-deisopropylation (M5), N,N-dealkylation (M6), and oxidation and dehydrogenation (M7). Only five metabolites (M2, M3, and M5-M7) were detected in human liver microsomes. In the conventional approach using extracted ion monitoring for values of mass increase or decrease by known metabolic reactions, only five metabolites (M1-M5) were found in rat liver microsomes, whereas three metabolites (M2, M3, and M5) were found in human liver microsomes. This study revealed that nontargeted metabolomics combined with high-resolution mass spectrometry and multivariate analysis could be a more efficient tool for drug metabolite identification than the conventional approach. These results might also be useful for understanding the pharmacokinetics and metabolism of DN203368 in animals and humans.

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