Preliminary concept paper



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2. Studies designed to identify drug metabolizing CYP enzymes


If human in vivo data or metabolic pathway identification studies conducted in vitro indicate CYP enzymes contribute >25% of a drug’s clearance, studies to identify drug metabolizing CYP enzymes in vitro are recommended. This recommendation includes cases in which oxidative metabolism is followed by transferase reactions, because a drug-drug interaction that inhibits oxidation of the parent compound can result in elevated levels of the parent compound.
a) General experimental methods for identifying drug metabolizing CYP enzymes
There are four well characterized methods for identifying the individual CYP enzymes responsible for a drug’s metabolism. The respective methods use 1) specific chemical inhibitors; 2) individual human recombinant CYP enzymes, 3) antibodies as specific enzyme inhibitors; 4) a bank human liver microsomes characterized for CYP activity that were prepared from individual donor livers. At least two of the methods should be performed to identify the specific enzyme(s) responsible for a drug’s metabolism.
Either pooled human liver microsomes or microsomes prepared from individual liver donors may be used for the methods a.1 and a.3. For correlation analysis (a.4), a bank of characterized microsomes from individual donor livers is required.
Experiments to identify the CYP enzymes responsible for a drug’s metabolism should be conducted, whenever possible, with pharmacologically relevant concentrations of drugs. It is recommended that enzyme identification experiments be conducted under initial rate conditions (linearity of metabolite production rates with respect to time and enzyme concentrations). In some cases the experiments may be conducted under nonlinear conditions due to analytical sensitivity; results of these experiments should be interpreted with caution. Thus, reliable analytical methods should be developed to quantitate each metabolite produced by individual CYP enzymes selected for identification. For racemic drugs, individual isomers should be evaluated separately.
b) Considerations regarding the use of specific chemical inhibitors to identify drug metabolizing CYP enzymes
Most chemical inhibitors are not absolutely specific for an individual CYP enzyme, but a valuable attribute of chemical inhibitors is their commercial availability. Although not all inclusive, the chemical inhibitors listed in Table 2 can be used to identify individual CYP enzymes responsible for a drug’s metabolism and determine the relative contribution of an individual CYP enzyme.

Table 2: Chemical inhibitors for in vitro experiments



CYP

Inhibitor (1)


Preferred


Ki

(µM)


Inhibitor (1)

Acceptable

Ki


(µM)


1A2

furafylline (2)


0.6-0.73

-naphthoflavone

0.01

2A6

tranylcypromine

methoxsalen (2)



0.02-0.2

0.01-0.2


pilocarpine

tryptamine



4

1.7 (3)



2B6





3-isopropenyl-3-methyl diamantane (4)

2-isopropenyl-2-methyl adamantane (4)

sertraline

phencyclidine

triethylenethiophosphoramide (thiotepa)

clopidogrel

ticlopidine


2.2

5.3


3.2 (5)

10

4.8



0.5

0.2


2C8

quercetin

1.1

trimethoprim

gemfibrozil

rosiglitazone

pioglitazone



32

69-75


5.6

1.7


2C9

sulfaphenazole

0.3

fluconazole

fluvoxamine

Fluoxetine


7

6.4-19


18-41

2C19







ticlopidine

nootkatone



1.2

0.5


2D6

quinidine

0.027-0.4







2E1







diethyldithiocarbamate

clomethiazole

diallyldisulfide


9.8-34

12

150



3A4/5

ketoconazole

itraconazole



0.0037- 0.18

0.27, 2.3



troleandomycin

verapamil



17

10, 24


  1. Substrates used for inhibition studies include: CYP1A2, phenacetin-o-deethylation, theophylline-N-demethylation; CYP2A6, coumarin-7-hydroxylation; CYP2B6, 7-pentoxyresorufin-O-depentylation, bupropion hydroxylation, 7-ethoxy-4-(trifluoromethyl)-coumarin O-deethylation, S-mephenytoin-N-demethylation; Bupropion-hydroxylation; CYP2C8, taxol 6-alpha-hydroxylation; CYP2C9, tolbutamide 4-methylhydroxylation, S-warfarin-7-hydroxylation, phenytoin 4-hydroxylation; 2CYP2C19, (S)-mephenytoin 4-hydroxylation CYP2D6, dextramethorphan O-demethylation, desbrisoquine hyddroxylase; CYP2E1, chlorzoxazone 6-hydroxylation, aniline 4-hydroxylase; CYP3A4/5, testosterone-6ß-hydroxylation, midazolam-1-hydroxylation; cyclosporine hydroxylase; nefedipine dehydrogenation.

  2. Furafylline and methoxsalen are mechanism-based inhibitors and should be preincubated before adding substrate.

  3. cDNA expressing microsomes from human lymphoblast cells.

  4. Supersomes, microsomal isolated from insect cells transfected with baculovirus containing CYP2B6.

  5. IC50 values

The effectiveness of competitive inhibitors is dependent on concentrations of the drug and inhibitor. Experiments designed to identify and to quantitate the relative importance of individual CYP enzymes mediating a drug’s metabolism should use drug concentrations ≤Km. The experiments should include the inhibitor at concentrations that ensure selectivity and adequate potency. It is also acceptable to use a range of inhibitor concentrations.


Noncompetitive and mechanism-based inhibitors are not dependent on the drug (substrate) concentration. When using a mechanism-based inhibitor, it is necessary to pre-incubate the inhibitor for 30 minutes.
For additional information concerning inhibition experiments see Inhibition Section.
c) Considerations regarding the use of recombinant enzymes to identify drug metabolizing CYP enzymes
When a drug is metabolized by only one recombinant human CYP enzyme, interpretation of the results is relatively straightforward. However, if more than one recombinant CYP enzyme is involved, measurement of enzyme activity alone does not provide information concerning the relative importance of the individual pathways.
Recombinant CYP enzymes are not present in their native environment and are often over expressed. Accessory proteins (NADPH-CYP reductase and cytochrome b5) or membrane lipid composition may differ from native microsomes. Several approaches have been reported to quantitatively scale metabolic activity obtained using recombinant CYP enzymes to activities expected in the human liver microsomes; however, these methods have not been validated and their results are suspect.
d) Considerations regarding the use of specific antibodies to identify drug metabolizing CYP enzymes
The inhibitory effect of an inhibitory antibody should be tested at sufficiently low and high concentrations to establish the titration curve. If only one CYP enzyme is involved in the drug’s metabolism, > 80% inhibition is expected in a set of pooled or individual microsomes. If the extent of inhibition is low, it is difficult to determine whether the partial inhibition is due to the involvement of other CYPs in metabolism of the drug or the antibody has poor potency.
e) Considerations regarding the use of correlation analyses to identify drug metabolizing CYP enzymes
This approach relies on statistical analyses to establish a correlation between the production rate of an individual metabolite and activities determined for each CYP enzyme in a set of microsomes prepared from individual donor livers.
The set of characterized microsomes should include microsomes prepared from at least 10 individual donor livers. The variation in metabolic activity for each CYP enzyme should be sufficient between individual donor livers to ensure adequate statistical power. Enzyme activities in the set of microsomes used for correlation studies should be determined using appropriate probe substrates and experimental conditions.
Results are suspect when a single outlying point dictates the correlation coefficient. If the regression line does not pass through or near the origin, it may indicate that multiple CYP enzymes are involved or reflect a set of microsomes that are inherently insensitive.

Appendix B. Evaluation of CYP inhibition


A drug that inhibits a specific drug-metabolizing enzyme can decrease the metabolic clearance of a co-administered drug that is a substrate of the inhibited pathway. A consequence of decreased metabolic clearance is elevated blood concentrations of the coadministered drug, which may cause adverse effects or enhanced therapeutic effects. Also, the inhibited metabolic pathway could lead to decreased formation of an active compound, resulting in decreased efficacy.
1. Probe substrates
In vitro experiments that are conducted to determine whether a drug inhibits a specific CYP enzyme involve incubation of the drug with probe substrates for the CYP enzymes.
There are two scientific criteria for selection of a probe substrate - the substrate should be selective (predominantly metabolized by a single enzyme in pooled human liver microsomes or recombinant P450s) and should have a simple metabolic scheme (ideally no sequential metabolism). There are also some practical criteria- commercial availability of substrate and metabolite(s); assays that are sensitive, rapid, and simple; and a reasonable incubation time.
Preferred substrates listed in Table 3 meet a majority of the criteria listed above. Acceptable substrates meet some of the criteria, and are considered acceptable by the scientific community.
Table 3. Preferred and acceptable chemical substrates for in vitro experiments

CYP

Substrate

Preferred



Km

(µM)


Substrate

Acceptable



Km

(µM)


1A2

phenacetin-O-deethylation

1.7-152

7-Ethoxyresorufin-O-deethylation

Theophylline-N-demethylation

Caffeine-3-N-demethylation

Tacrine 1-hydroxylation



0.18-0.21

280-1230


220-1565

2.8, 16


2A6

coumarin-7-hydroxylation

nicotine C-oxidation



0.30-2.3

13-162








2B6

Efavirenz hydroxylase


17-23


Propofol hydroxylation

S-mephenytoin-N-demethylation

Bupropion-hydroxylation


3.7-94

1910


67-168

2C8

Taxol 6-hydroxylation

5.4-19

Amodiaquine N-deethylation

Rosiglitazone para-hydroxylation



2.4,

4.3-7.7


2C9

tolbutamide methyl-hydroxylation

S-warfarin 7-hydroxylation

diclofenac 4’-hydroxylation


67-838

1.5-4.5


3.4-52

Flurbiprofen 4’-hydroxylation

Phenytoin-4-hydroxylation




6-42

11.5-117


2C19

S-mephenytoin 4’-hydroxylation

13-35

Omeprazole 5-hydroxylation

Fluoxetine O-dealkylation



17-26

3.7-104


2D6

()-bufuralol 1’-hydroxylation

dextromethorphan O-demethylation



9-15

0.44-8.5


Debrisoquine 4-hydroxylation


5.6

2E1

chlorzoxazone 6-hydroxylation



39-157

p-nitrophenol 3-hydroxylation

Lauric acid 11-hydroxylation

Aniline 4-hydroxylation


3.3

130


6.3-24

3A4/5*

midazolam 1-hydroxylation

testosterone 6-hydroxylation




1-14

52-94


Erythromycin N-demethylation

Dextromethorphan N-demethylation

Triazolam 4-hydroxylation

Terfenadine C-hydroxylation

Nifedipine oxidation


33 – 88

133-710


234

15

5.1- 47




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