Metabolism

Contract Research | Absorption/Transport | Metabolism | Induction | Physico-Chemical | Toxicity | Sponsor-Initiated Protocols

Metabolic Stability and Metabolite Profiling
Metabolic stability influences both oral bioavailability and half life (Xenobiotica 31:591 [2001])¹. For example, with cytochrome P450 substrates of low and moderate in vivo clearance, there is a good correlation between in vitro metabolic stability and in vivo clearance (Biochem. Pharmacol.47:1469 [1994])². BD Biosciences provides screening for in vitro metabolic stability. This test uses hepatocytes, pooled liver microsomes, S9 (human and/or preclinical species) or BD Supermix™ cDNA-expressed enzymes with appropriate positive and negative controls. Assessment of both phase 1 and phase 2 enzyme metabolism is available. A standard set of substrate concentrations and incubations may be used. Metabolism is measured by loss of parent compound. Metabolite profiling is also available. HPLC analysis with absorbance, fluorescence , radiometric or mass spectrometric detection is available. Alternatively, the incubations can be returned to the sponsor for analysis.

High Throughput Cytochrome P450 Inhibition Screen
The majority of drug-drug interactions are metabolism-based and of these, most involve cytochrome P450 (Drug-Drug Interactions: Scientific and Regualtory Perspectives pp7-35 [1999]; Metabolic Drug Interactions pp3-19 [2000])^3,4. For example, if a new chemical entity is a potent cytochrome P450 inhibitor, it may inhibit the metabolism of a co-administered medication, potentially leading to adverse clinical events. To expedite identification of drug candidates with cytochrome P450 inhibitory potential, we offer a high throughput screening service. The inhibition of human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and other isoforms are assessed using BD Supersomes™ Enzymes as enzyme sources and the fluorescence detection method described in publications from BD Biosciences (J. Pharmarcol. Toxicol. Methods 44:325 [2000]^5,6; Meth. Enzymol. 357:276 [2002]) and others ( Drug Metab. Dispos. 27:436 [1999])^7. Tests are conducted in 96-well microplates and may use the following fluorescent P450 substrates: resorufin benzylether, 3-cyano-7-ethoxycoumarin, ethoxyresorufin, 7-methoxy-4-trifluoromethyl -coumarin, 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin, 7-benzyloxyquinoline, dibenzyfluorescein or 7-benzyloxy-4-trifluoromethylcoumarin. Data are reported as IC50 values or percent inhibition when using only one or two concentrations of test compound.

Inhibition of Cytochrome P450 — IC50 Determination
This test uses cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 enzymes and several model substrates including phenacetin, coumarin, paclitaxel, diclofenac, (S)-mephenytoin, bufuralol, p-nitrophenol, testosterone, nifedipine and midazolam. Other enzyme substrate pairs may be available. Because inhibition constants are substrate dependent for CYP3A4, multiple substrates (e.g. testosterone, nifedipine and midazolam) are available for this enzyme. This protocol uses a single substrate concentration near the apparent Km and multiple test article concentrations (number and spacing are flexible). An IC50 is determined as the point where 50% inhibition of enzyme catalytic activity occurs. Enzymes can be tested together, individually or in groups. There are several advantages to using cDNA-expressed enzymes for inhibition studies (Adv.Pharmacol.43:171 [1997])⁸. For example, IC50 values obtained can be compared with clinically significant inhibitors of the same enzyme without the complication of competing pathways of metabolism. Protocols employing human liver microsomes (HLM) as the source of enzyme are also available.

Inhibition of Cytochrome P450 — Ki Determination
This test uses cDNA-expressed CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes and the same model substrates used with IC50 determinations. This protocol uses multiple substrate concentrations near the apparent Km and multiple inhibitor concentrations (number and spacing are flexible). Choice of the inhibitor concentrations requires a prior IC50 determination. A Ki is calculated by Dixon plots.

Inhibition of CYP3A4 Catalytic Activity
The CYP3A4 enzyme is significantly involved in the metabolism of more than half of commercially available drugs. Consequently, CYP3A4 is frequently associated with metabolism-based drug-drug interactions. Obtaining a clear CYP3A4 inhibition profile may be crucial to the successful development of your leads. We offer multiple CYP3A4 catalytic activity assays to assess substrate dependence of IC50 values, activation and the complex inhibition kinetics associated with this enzyme (Drug Metab. Dispos. 28:1440 [2000]; Drug Metab. Dispos. 28:360 [2000])^9,10. High-throughput assays conducted in 96-well microplates using fluorescence detection with up to four different CYP3A4 substrates (BzRes, BQ, BFC, DBF) are available. In addition, assays using the classical substrate testosterone, as well as two clinically relevant compounds, midazolam and nifedipine, are available.

Inhibition of UDP-glucuronosyl transferase (UGT) — IC50 Determination
We can test for the inhibition of several UGT isoforms including UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7 enzymes using the model substrates 7-hydroxy-trifluoromethylcoumarin, 17beta-estradiol, trifluoperazine or bilirubin. For additional UGT isoforms, please inquire. This protocol uses a single substrate concentration near the apparent Km and multiple test article concentrations (number and spacing are flexible). An IC50 value is determined as the point where 50% inhibition of enzyme catalytic activity occurs. These enzymes can be tested together, individually or in groups. Together, these enzymes represent the major human drug-metabolizing UGTs (Annu. Rev. Pharmacol. Toxicol. 40:581 [2000])¹¹. There are several advantages to using cDNA-expressed enzymes for inhibition studies. For example, IC50 values obtained can be compared with clinically significant inhibitors of the same enzyme without the complication of competing pathways of metabolism.

Cytochrome P450 Reaction Phenotyping
The number and identity of cytochrome P450 enzymes responsible for the metabolism of a drug affects population variability in metabolism. Which enzyme or enzymes are capable of metabolizing your test compound? Reaction phenotyping typically involves use of liver microsomes with enzyme-selective chemical or antibody inhibitors and a panel of cDNA-expressed enzymes to provide evidence of the number and identity of enzymes involved in the metabolism of the substrate. The amount of each cDNA-expressed enzyme is chosen to be proportional to the activity of the same enzyme in pooled HLMs. Protein concentration is standardized by the addition of control microsomes (without cytochrome P450 enzymes). Metabolism is measured by loss of parent compound and/or formation of metabolites. Multiple protocols are available to meet your needs (e.g. discovery vs. development). Additional tests are available to determine the involvement of principal non-cytochrome P450 enzymes (e.g. flavin-containing monooxygenase). HPLC analysis with absorbance, fluorescence, radionetric or mass spectrometric detection is available. Alternatively, the incubations can be returned to the sponsor for analysis. The importance of reaction phenotyping has been recently reviewed (Xenobiotic 28:1167[1999];Biochem. Pharmacol. 57:465[1999])^12,13.

Mechanism-based or "suicide" Inhibitor Testing
Compounds that fail in the development stage because of toxicity are often found to be mechanism-based inhibitors or so-called "suicide inhibitors" of cytochrome P450 (Drug-Drug Interactions pp387-414 [2002])¹⁴. Anticipate these problems long before valuable resources have been devoted to compounds that eventually may fail. This test determines the NADPH and time-dependent loss of catalytic activity due to the test compound in liver microsomes or cDNA-expressed enzymes employing various substrates and procedures

High Throughput Aromatase (CYP19) Inhibition Screen
Human aromatase (CYP19) converts C19 androgens to aromatic C18 estrogenic steroids and also metabolizes some xenobiotics. Inhibitors of this enzyme can be used therapeutically to treat postmenopausal breast cancer and other estrogen-dependent diseases. Drugs and other environmental chemicals exhibiting undesired aromatase inhibition have been implicated as endocrine disrupting agents. We would test for inhibition in microplates using the fluorescent substrate dibenzyl fluorescein and cDNA-expressed CYP19 (Anal. Biochem. 284:427 [2000])¹⁵. Data are reported as IC50 values or percent inhibition when using only one or two concentrations of test compound.

Service Information/References

• Cytochrome P450 Reaction Phenotyping [ PDF ]
• Inhibition of Cytochrome P450 and UGT [ PDF ]
• Metabolic Stability [ PDF ]

1. Worboys, P.D. and Carlile, D.J. Implications and consequences of enzyme induction on preclinical and clinical development. Xenobiotica 31:539-556 [2001].
2. Houston, J.B. Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. Biochem. Pharmacol. 47:1469 [1994].
3. Guengerich, F. P. Role of cytochrome P450 enzymes in drug-drug interactions. Drug-drug interactions: scientific and regulatory perspectives. Li AP (ed.) Academic Press, San Diego pp7-35 [1997].
4. Thummel, K.E., Kunze, K.L. and Shen, D.D. Metabolically-based drug-drug interactions: Principles and mechanisms. Metabolic Drug Interactions . Lippincott, Williams & Wilkins, Philadelphia, pp3-19 [2000].
5. Crespi, C.L. and Stresser, D.M. Fluorometric Screening for Metabolism-Based Drug-Drug Interactions
   J. Pharmacol. Toxicol. Method. 44:325 [2000].
6. Crespi, C.L., Miller, V.P. and Stresser, D.M. Design and application of fluorometric assays for human cytochrome P450 inhibition. Meth. Enzymol. 3 5 7:276 [2002].
7. Favreau, L.V., Palamanda, J.R., Lin, C.C. and Nomeir A.A. Improved Reliability of the Rapid Microtiter Plate Assay Using Recombinant Enzyme in Predicting CYP2D6 Inhibition in Human Liver Microsomes. Drug Metab. Dispos. 27:436 [1999].
8. Crespi, C.L. and Penman, B.W. Use of cDNA-expressed human cytochrome P450 enzymes to study potential drug-drug interactions. Advances in Pharmacology 43:171 [1997].
9. Stresser, D.M., Turner, S.D., Blanchard, A.B., Miller, V. P., Erve, J.C.L., Dandeneau, A.C. and Crespi, C.L. Substrate-dependent modulation of CYP3A4 catalytic activity: analysis of 27 test compounds with four fluorometric substrates. Drug Metab. Dispos. 28:1440 [2000].
10. Wang, R.W., Newton, D.J., Liu, N., Atkins, W.M. and Lu, A.Y. Human cytochrome P-450 3A4: in vitro-drug-drug interaction patterns are substrate-dependent .Drug Metab. Dispos.28(3):360 [2000].
11. Tukey, R.H. and Strassburg, C.P. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu. Rev. Pharmacol.Toxicol.40:581 [2000].
12. Clarke, S.E. in vitro assessment of human cytochrome P450. Xenobiotica 28:1167 [1999].
13. Rodrigues, A.D. Integrated cytochrome P450 reaction phenotyping: Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochem. Pharmacol. 57:465
    [1999].
14. Jones, D.R. and Hall, S.D. Mechanism-based inhibition of human cytochromes P450: In vitro kinetics and in vitro-in vivo correlations Drug-Drug Interactions, Martel Dekker, New York, pp387-414 [2002].
15. Stresser, D.M., Turner, S.D., McNamara, J., Stocker, P., Miller, V.P. , Crespi, C.L. and Patten, C.J. A high-throughput screen to identify inhibitors of aromatase (CYP19). Anal. Biochem. 284:427[2000].

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The complete FDA guidance document can be viewed on the web at:
http://www.fda.gov/cder/guidance/clin3.pdf