B

Figure 5.2. Concentration- and time-dependent inactivation. (a) natural log of percentage of activity remaining versus time. (b) the corresponding l/W (min"1)

Figure 5.2. Concentration- and time-dependent inactivation. (a) natural log of percentage of activity remaining versus time. (b) the corresponding k _ kinact X [/] /r ,ni kobS - + [I] (5'10)

An IC50 shift assay. Determination of kinact and KI is not suitable for discovery-stage screening purposes, since it is resource intensive and time consuming. In IC50, shift methodology is proposed where different [I] are incubated for 30 min with enzyme in the presence or absence of NADPH (Obach et al. 2007). An aliquot of each sample is then transferred to a second incubation with a probe substrate. IC50 values are determined for each condition (+/—NADPH) by plotting percent activity of the control versus [I]. If the IC50 is decreased (i.e., shifted) in the presence of NADPH, the compound exhibits TDI. The new IC50 correlates well with kinact/KI (Fig. 5.3).

Figure 5.3. IC50 shift graph: percent activity versus inhibitor concetration. Assay is run in presence and absence of NADPH after preincubation period.

+NADPH

0.001

-NADPH

Hepatocyte TDI studies may be a useful tool for considering a system with broader metabolically competent processes (Zhao et al. 2005; McGinnity et al. 2006).

See Table 5.5.

Figure 5.2 Continued. double reciprocal plot of the rates of inactivation as a function of inhibitor concentration. kinact and KI are obtained from the reciprocal of y-intercept and negative x-intercept, respectively.

Table 5.5. Compounds that are mechanism-based inhibitors of P450 isoforms

P450 Isoform

Mechanism-based inhibitors

CYP1A2 Clorgyline, oltipraz, resveratrol, rofecoxib, zileuton

CYP2B6 Bergamottin, Clopidogrel, ThioTEPA, ticlopidine

CYP2C8 Amiodarone, fluoxetine, gemfibrozil glucuronide, isoniazid, nortriptyline, phenelzine, verapamil CYP2C9 Tienilic acid

CYP2C19 ThioTEPA, ticlopidine

CYP2D6 Paroxetine, serpentine

CYP3A4/5 Bergamottin, diltiazem, erythromycin, fluvoxamine, mifepristone, ritonavir, saquinavir, troleandomycin

Furanocoumarin derivatives present in grapefruit juice are mechanism-based inhibitors of CYP3A4 in the intestine and could lead to DDI.

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