Phase I Drug Metabolism

By: Robert Gates, Biofiles Volume 6 Article 1

Phase I biotransformation reactions introduce or expose functional groups on the drug with the goal of increasing the polarity of the compound. Although Phase I drug metabolism occurs in most tissues, the primary and first pass site of metabolism occurs during hepatic circulation. Additional metabolism occurs in gastrointestinal epithelial, renal, skin, and lung tissues. Within cells, most phase I enzymes are located in the endoplasmic reticulum and thus are enriched in microsomal preparations.

Phase I reactions are broadly grouped into three categories, oxidation, reduction, and hydrolysis. As most small molecule drugs are lipophilic in nature, drug metabolism converts these hydrophobic compounds into more water soluble compounds that can be excreted. Typically, oxidation is the most common phase I reaction. The hepatic cytochrome P450 system is the most important of the phase I oxidation systems (Figure 1).

Figure 1. Our hepatic microsomal cytochrome P450 enzymes are membrane-bound oxygenases containing a thiolate heme near the active site. Electron donation for the oxidatve mechanism is supplied from a coupled NAD(P)H reductase and cytochrome b5 system. Until recently, the difficulty of isolating and reconstituting native enzymes from human tissue has hampered their use for studies with well-defined in vitro drug metabolism systems. For this reason, multienzyme microsomal preparations are often utilized for the elucidation of the oxidate fate of drugs and other xenobiotics. Our recombinant microsomal preparations have the advantage of a single cytochrome P450 isozyme activity.

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Cytochrome P450 Enzymes

The cytochrome P450s are members of a superfamily of monoxygenases that catalyze the oxidative metabolism of xenobiotics, the initial step in the biotransformation and elimination of a wide variety of drugs and environmental pollutants. P450s are located primarily in the endoplasmic reticulum of liver tissue.

Cytochrome P450 Enzymes

Cerosomes: P450 Microsomes Expressed in Yeast

Use of a eukaryotic, yeast host allows efficient expression of the active isozyme with more accurate post-translational modification. Cerosomes are prepared from Saccharomyces cervisiae expressing the human cytochrome P450 isozyme and human NADPH reductase and cytochrome b5 in some preparations.

The eukaryotic system provides an isozyme system with the following advantages:

  • Recombinant isozyme mimics the active, native human enzyme
  • Higher specific activity and better signalto- noise ratio (Figure 2)
  • Consistent and reproducible results

Figure 2. Specific activity determined by a fluorescent product generated from an enzyme-specific non-fluorescent substrate after incubation with cytochrome P450.The fluorescent emission vs. time yields activity per mg protein.

The Cerosomes cytochrome P450 isozymes are supplied in a solution containing 50 mM potassium phosphate, pH 7.4, 5% glycerol, 1 mM EDTA, and 1 mM PMSF.

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Cytochrome Reductase and Cytochrome b5

The oxidative mechanism of the cytochrome P450 system requires an electron transport system of proteins to support catalysis. Cytochrome P450 (NADP) reductase is coexpressed with all of our recombinant cytochrome P450 preparations. Cytochrome b5 is also coexpressed with some preparations.

Cytochrome P450 reductase catalyzes the reduction of hemethiolate-dependent monooxygenases such as EC (unspecified xenobiotic monooxygenases2) and is part of the microsomal hydroxylating system. This reductase is a flavoprotein containing FMN and FAD. It also reduces cytochrome b5 and cytochrome c.

Cytochrome b5 is a 15 kDa membranebound flavoprotein capable on enhancing the redox potential of the P450 oxidative system.

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Selected Cytochrome P450 Substrates

Name Description Cat. No.
Debrisoquine sulfate Its metabolite may induce Parkinsonism. D1306-100MG
3-Oxo-1-cyclopentanecarboxylic acid Substrate used in a study of biohydroxylation with mutants of cytochrome P450 BM-3. 550485-1G
Resorufin benzyl ether - B1532-5MG
Resorufin ethyl ether - E3763-1MG
Resorufin methyl ether Fluorimetric substrate for cytochrome P450 linked enzymes. M1544-1MG
Resorufin pentyl ether Fluorimetric substrate for cytochrome P450 linked enzymes, CYP2B and CYP2B4. P0928-1MG

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Selected Antibodies to Phase I Enzymes

Product Name Host Clone No. Form Gene Symbol Species Reactivity Application Prestige Antibody Cat. No.
Monoclonal Anti-AKAP9 mouse 1A6 purified immunoglobulin AKAP9, human human ELISA (i) WB - WH0010142M1-50UG
Anti-AKAP9/AKAP450/ CG-NAP goat - affinity isolated antibody AKAP9, human human mouse ELISA (i) IHC WB - SAB2500052-100UG
Anti-Aromatase rabbit - affinity isolated antibody CYP19A1, human human IF (i) WB - A7981-25UL
Anti-Aromatase/CYP19A1 goat - affinity isolated antibody CYP19A1, human human ELISA (i) WB - SAB2500110-100UG
Anti-CYP11A1 rabbit - affinity isolated antibody CYP11A1, human human IHC (p) PA Yes HPA016436-100UL
Anti-CYP17A1 goat - affinity isolated antibody CYP17A1, human canine cat human ELISA (i) WB - SAB2500285-100UG
Anti-CYP1A1 (AB2) rabbit - affinity isolated antibody CYP1A1, human human WB - AV41404-50UG
Anti-CYP1A1 (AB3) rabbit - affinity isolated antibody CYP1A1, human human WB - AV41405-50UG
Anti-CYP1B1 rabbit - affinity isolated antibody CYP1B1, human human WB - AV51761-50UG
Monoclonal Anti-CYP1B1 mouse 2F8 purified immunoglobulin CYP1B1, human human ELISA (i) - WH0001545M3-100UG
Anti-CYP24A1 rabbit - affinity isolated antibody CYP24A1, human human IHC (p) PA Yes HPA022261-100UL
Monoclonal Anti-CYP24A1 mouse 1F8 purified immunoglobulin CYP24A1, human human ELISA (i) WB - WH0001591M7-100UG
Anti-CYP26A1 (481-495) rabbit - IgG fraction of antiserum CYP26A1, human human WB - C6498-200UL
Anti-CYP26B1 rabbit - affinity isolated antibody CYP26B1, human human IHC (p) PA WB Yes HPA012567-100UL
Anti-CYP2C19 rabbit - affinity isolated antibody CYP2C19, human human IHC (p) PA WB Yes HPA015066-100UL
Anti-CYP2C8 rabbit - affinity isolated antibody CYP2C8, human human IHC (p) PA WB Yes HPA013547-100UL
Anti-CYP2D6 rabbit - IgG fraction of antiserum CYP2D6, human human IHC WB - AV41675-100UG
Anti-CYP2E1 rabbit - affinity isolated antibody CYP2E1, human human IHC (p) PA WB - HPA009128-100UL
Anti-CYP2E1 rabbit - affinity isolated antibody CYP2E1, human human WB - AV41424-50UG
Monoclonal Anti-CYP2J2 mouse 2D10 purified immunoglobulin CYP2J2, human human ELISA (i) WB - WH0001573M1-100UG
Anti-CYP3A4 mouse - IgG fraction of antiserum CYP3A4, human human ELISA (c) IHC (p) WB - SAB1400064-50UG
Anti-CYP3A4 rabbit - IgG fraction of antiserum CYP3A4, human human ELISA (c) WB - SAB1400065-100UG
Anti-CYP4F2 rabbit - affinity isolated antibody CYP4F2, human human IHC (p) PA Yes HPA014048-100UL
Anti-CYP51A1 (495-509) rabbit - IgG fraction of antiserum CYP51A1, human human WB - C8873-200UL
Anti-CYP7A1 rabbit - affinity isolated antibody CYP7A1, human human IHC (p) PA WB Yes HPA010970-100UL
Anti-CYP7B1 rabbit - affinity isolated antibody CYP7B1, human human IHC (p) PA WB Yes HPA017761-100UL
Monoclonal Anti-Cytochrome P450 1A2 mouse 3B8C1 purified immunoglobulin CYP1A2, human Cyp1a2, rat human rat IHC WB - C5118-25UL
Monoclonal Anti-Cytochrome P450 2C6 (CYP2C6) mouse K1 purified immunoglobulin Cyp2c6, rat human rat ELISA (i) WB - C0746-25UL
Monoclonal Anti-Cytochrome P450 3A5 mouse F18 P3 B6 purified immunoglobulin CYP3A5, human human ELISA (i) IHC WB - C4743-25UL
Anti-DBH rabbit - affinity isolated antibody DBH, human human IHC (p) PA Yes HPA005960-100UL
Anti-FMO1 rabbit - affinity isolated antibody FMO1, human human IHC (p) PA Yes HPA023680-100UL
Anti-FMO3 rabbit - affinity isolated antibody FMO3, human human IHC (p) PA WB Yes HPA013750-100UL
Anti-KMO rabbit - affinity isolated antibody KMO, human human IHC (p) PA Yes HPA031115-100UL
Anti-PAM rabbit - affinity isolated antibody PAM, human human WB - SAB2101711-50UG
Anti-POR rabbit - affinity isolated antibody POR, human human IHC (p) PA WB Yes HPA010136-100UL
Anti-TPH1 rabbit - affinity isolated antibody TPH1, human human IHC (p) PA WB Yes HPA022483-100UL
Anti-YWHAZ rabbit - IgG fraction of antiserum YWHAZ, human human WB - AV48046-100UG

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